Stable formulation of silk-derived proteins

Abstract

Biotherapeutic eye drops may contain silk-derived proteins as active ingredients. Ophthalmic formulations are essential for dosage form delivery, user requirements, and maintaining product stability. The formulations described herein provide user-friendly ophthalmic solutions while maintaining product stability even after extended storage. Numerous excipients, manufacturing processes, and container closures were evaluated for their ability to stabilize silk-derived proteins under ambient and accelerated conditions. Analysis showed that a small subset of protein-containing formulations met the high physicochemical property standards required for therapeutic ophthalmic solutions.

Description

[Technical field]

[0001] Related Applications This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 094,748, filed October 21, 2020, No. 63 / 094,709, filed October 21, 2020, and No. 62 / 936,294, filed November 15, 2019, which applications are incorporated herein by reference.

[0002] government support This invention was made with Government support under Grant No. W81XWH-17-C-0147 awarded by the U.S. Army. The Government has certain rights in this invention. [Background technology]

[0003] 2. Background of the Invention Peptide and protein therapeutics are becoming increasingly popular as treatments for a variety of diseases. Historical approaches to isolating these molecules included harvesting them from animal organs and tissues. However, the recent success of recombinant DNA technology has spurred the development of new protein biotherapeutics over the past two decades. Peptides, proteins, and protein-based biosimilars offer many advantages in treating diseases compared to chemically synthesized therapeutics. For example, purified antibodies have highly specific secondary and tertiary folding patterns underlying their structure and remain functional after being administered to patients. Similarly, therapeutic peptides used to stimulate or inhibit intracellular signaling (hormones, blood clotting factors, etc.) are potent, fast-acting, and metabolized utilizing common proteolytic pathways in the host.

[0004] The efficacy of peptides and proteins depends on their ability to uniquely and effectively interface with targets such as cell surface receptors, lipid rafts, or intracellular / extracellular molecules. This specificity requires that therapeutic peptides and proteins maintain a functional organization of amino acids and amino acid conformations that form higher-order secondary structures (e.g., α-helical, β-sheet), tertiary structures (three-dimensional shapes), and quaternary structures (interactions of multiple protein subunits). These arrangements are directed by electrostatic interactions between amino acid residues, including covalent (e.g., disulfide bonds) and non-covalent (e.g., hydrogen, hydrophobic, ionic) bonds, all of which are dependent on the surrounding environmental parameters governed by physiological homeostasis to facilitate these bindings.

[0005] Small changes in the pH of tissues or solutions can alter the concentration of hydrogen ions, promoting or inhibiting the protonation of amino acid residues, thereby attracting or repelling adjacent amino acids with opposite or similar charges, respectively. Similarly, an increase in temperature of tissues or solutions containing proteins can increase the internal energy, destabilizing the proteins through peptide hydrolysis and rearrangement of protein structure. Thus, there is a dire need to control and maintain the environment to maintain the efficacy of peptide and protein formulations.

[0006] The human body has numerous processes and mechanisms to maintain homeostasis, from ion channels and proton pumps at the cellular level, to the collective function of each organ in the body, to the systemic vascular and lymphatic systems to resolve fluid waste gradients in these organs and tissues. However, when proteins are dislodged from these feedback-driven safeguards, they are vulnerable to compromise or loss of functionality. This is particularly true for therapeutic peptides and proteins, whose functional structure can be rapidly degraded by ambient temperatures and non-physiological solution conditions. Protein conformational changes can be due to the formation or cleavage of covalent bonds (chemical instability), interactions with neighboring proteins, or solution additives that impair solubility.

[0007] Chemical instability of proteins is commonly caused by oxidation (e.g., by UV irradiation, and / or the presence of peroxides or metal ions) or amino acid deamidation induced by changes in pH or elevated temperature. These latter changes in solution conditions can lead to protein aggregation and reduced protein solubility that can result from mechanical (e.g., shear) and interfacial stresses imposed on dissolved proteins in aqueous solutions. Therefore, the design of protein-containing formulations must address these stressors as much as possible to facilitate shelf-stable therapeutics and maintain efficacy.

[0008] Currently, the primary strategy for therapeutic peptide and protein stability is to formulate solutions that mimic the physiological environment of tissues. Salt-based buffer systems are commonly used to prevent large fluctuations in solution pH that occur over time (e.g., with the absorption of carbon dioxide, which acidifies the solution) or with peptide hydrolysis. Excipients are used to increase the osmolality of the solution and reduce the chance of protein-protein interactions and aggregation. Similarly, the addition of surfactants is used to reduce the possibility of interfacial stress and physical instability. Alternatively, many protein solutions are stored at refrigerated temperatures to extend shelf life, but this is not ideal when the therapeutic is administered routinely or multiple times per day. Finally, some protein therapeutics are stored as lyophilized powders to minimize protein degradation. These solutions are solubilized immediately prior to administration, but variability in the volume of solvent used for solubilization can easily introduce errors in drug dosing. Summary of the Invention [Problem to be solved by the invention]

[0009] Thus, there is a need for a stable therapeutic protein formulation that is highly soluble in solution, has long-term stability and low particulate count, and is compatible with other readily available ingredients. There is also a need for a protein formulation for the treatment of eye-related conditions that can maintain the stability of the protein in solution for extended periods of time, thus increasing the shelf life and efficacy of the formulation. There is also a need for a formulation that can be used to treat eye-related conditions without the use of protein additives. The present disclosure meets these needs. [Means for solving the problem]

[0010] Summary of the Invention The present invention provides formulations for improving the physical and chemical stability of proteins such as modified silk fibroin. The silk-derived proteins (SDPs) described herein are protein compositions that have reduced beta-sheet activity, resulting in highly soluble materials. SDPs can be easily incorporated into solution-based product formulations at high concentrations. Another advantage is that SDPs are highly miscible with other soluble components, such as those typically included in therapeutic formulations.

[0011] While traditional agents used to pursue aqueous protein stability either do not affect or adversely affect the physical stability of the SDP in solution, the selection of specific components of the formulation described herein is unique: specific buffer salts, osmotic agents, and surfactants extend the stability of the SDP at room temperature without protein degradation or loss of protein efficacy.

[0012] The present disclosure provides a formulation comprising a fibroin-derived protein composition, the fibroin-derived protein composition having an average molecular weight of 15-35 kDa. The formulation also comprises a buffer, polysorbate-80, and one or more osmotic agents; the formulation has a pH of 4.5-6.0 and a particulate count of 50 mL or less after a storage period of 12 weeks or more, or 24 weeks or more, at 4° C.-40° C. for particulates having a diameter of 10 micrometers or more.

[0013] Further, the present disclosure provides a formulation comprising about 0.1% to about 3% by weight of silk-derived protein-4 (SDP-4); polysorbate-80, about 10 millimolar to about 50 millimolar acetate buffer, and an osmotic agent; wherein the formulation has a pH of 5.2 to 5.8, an osmolality of 175 mOsm / kg to 185 mOsm / kg, and a particulate count of 50 / mL or less for particulates having a diameter of 10 micrometers or greater after a storage period of 12 weeks or more, or 24 weeks or more at 40° C.

[0014] Particular embodiments include formulations comprising about 0.1% to about 3% by weight of silk-derived protein, where the silk-derived protein may have a primary amino acid sequence of the fibroin-derived protein that differs from native fibroin by at least 6% in terms of the absolute value of the sum of the differences in amino acid content of serine, glycine, and alanine, the cysteine ​​disulfide bonds between the fibroin heavy protein chain and the fibroin light protein chain of the fibroin-derived protein are reduced or eliminated, and the fibroin-derived protein contains more than 46% glycine amino acids and more than 30% alanine amino acids. The fibroin derived protein has a serine content that is reduced by 40% or more compared to native fibroin protein, such that the fibroin derived protein contains less than 8% serine amino acids; and the average molecular weight of the fibroin derived protein is about 15 kDa to about 35 kDa; and polysorbate-80, an acetate buffer of about 10 mM to about 50 mM, and an osmotic agent; wherein the formulation is characterized by having a pH of 5.2 to 5.8, an osmolality of 175 mOsm / kg to 185 mOsm / kg, and a particulate count of 50 mL or less after a storage period of more than 12 weeks at 4° C. to 40° C. for particulates having a diameter of 10 micrometers or greater.

[0015] In one embodiment, the buffer salt produces a solution pH of 5.5; the buffer salt used has a functional range between 3.7 and 5.6; the osmotic agent used produces a solution with an osmolality of 160-200 mOsm / kg; the osmotic agent used has a concentration of 0.5% wt. / wt; and the surfactant used has a concentration of 0.05-0.5% wt. / wt.

[0016] In another aspect, certain embodiments provide ophthalmic formulations that may be used to treat certain eye-related conditions, particularly to treat or alleviate other symptoms of dry eye disease.

[0017] Thus, preferred embodiments include ophthalmic formulations that may include about 0.04% to about 0.1% by weight polysorbate-80, about 0.2% to about 0.3% by weight sodium acetate, and an acetate buffer comprising about 0.01% to about 0.03% by weight acetic acid, and an osmotic agent comprising about 0.6% to about 0.9% by weight glucose and about 0.4% to about 0.9% by weight magnesium chloride, the formulation having a pH of 5.2 to 5.8 and an osmolality of 175 mOsm / kg to 185 mOsm / kg, and optionally including silk-derived protein.

[0018] One embodiment of the ophthalmic formulation consists essentially of about 0.04% to about 0.1% by weight polysorbate-80, an acetate buffer comprising about 0.2% to about 0.3% by weight sodium acetate, and about 0.01% to about 0.03% by weight acetic acid, and an osmotic agent comprising about 0.6% to about 0.9% by weight dextrose and about 0.6% to about 0.9% by weight magnesium chloride, wherein the formulation has a pH of 5.2 to 5.8 and an osmolality of 175 mOsm / kg to 185 mOsm / kg, and optionally includes a silk-derived protein.

[0019] In some embodiments, the ophthalmic formulations described herein further comprise a therapeutic protein or peptide composition. In certain embodiments, the wt% of the protein or peptide in the formulation is about 0.01% to about 15%. In certain embodiments, the wt% of the protein or peptide is about 0.1% to about 5%, or about 1% to about 3%. In a preferred embodiment, the protein is a hydrophobic protein. In one specific embodiment, the protein is SDP-4. In various embodiments, the wt% of SDP-4 in the formulation is about 0.01% to about 15%. In additional embodiments, the wt% of SDP-4 is about 0.1% to about 5%, or about 1% to about 3%, or about 0.1%, 1%, or 3%.

[0020] Accordingly, certain embodiments of ophthalmic formulations include one or more buffering agents, a surfactant, and one or more osmotic agents; wherein the formulation has a pH of 4.5 to 6.0, wherein the formulation maintains proteins in solution without gelling for a period of more than 4 weeks, and maintains a volume of 50 mL or less in terms of particulate count having a diameter of 10 micrometers or greater after a storage period of more than 12 weeks at 4° C. to 40° C.

[0021] In additional embodiments, the ophthalmic formulation may comprise one or more surfactants; one or more osmotic agents; and an acetate buffer system comprising about 0.1% to about 1.0% by weight sodium acetate and about 0.01% to about 0.1% by weight acetic acid, wherein the buffer system maintains the formulation at a pH of 4.5 to 6, wherein the formulation is capable of maintaining a protein in solution for a period of more than 4 weeks without gelling, and wherein the formulation is capable of maintaining a particulate count of 50 mL or less after a storage period of more than 12 weeks at 4° C. to 40° C. for particulates having a diameter of 10 μm or greater when the protein is added to the ophthalmic formulation. [Brief description of the drawings]

[0022] Brief description of the drawings The following drawings form part of the present specification and are included to further demonstrate certain embodiments or various aspects of the present invention. In some cases, embodiments of the present invention may be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and accompanying drawings may emphasize certain examples, or particular aspects of the invention. However, one of ordinary skill in the art will appreciate that some of the examples or aspects can be used in combination with other examples or aspects of the invention.

[0023] [Figure 1]Figure 1: Effect of temperature on physical stability of SDP-4. Graph summarizing solution particulate counts at 1.0% wt. / wt. SDP-4 solutions were maintained at defined temperatures and pH (30 min). No buffers or excipients were used. The figure shows subvisible particle counts by Coulter method after 4 weeks under the indicated solution conditions. Solutions held at 40°C showed increased particulate formation with increasing pH.

[0024] [Diagram 2] Figure 2: Effect of temperature on the physical stability of SDP-4. Graph showing summary of solution particle counts at 1.0% wt. / wt. SDP-4 solutions were maintained at defined temperatures and pH (200 min). No buffers or excipients were used. Coulter visible light particle counts are shown after 4 weeks under the solution conditions shown. Under all conditions, particle counts decreased significantly with increasing SDP-4 reaction time. At 40°C, particle generation increased with increasing pH.

[0025] [Diagram 3] Figure 3: Incubation time of SDP-4 affects physical stability. Summary graph of particle count (by Coulter method) at 1% wt / wt. SDP-4 solutions were incubated for 30 min (dark grey) or 200 min (light grey) and buffered with citric acid (CA) to indicate solution pH. Solutions were stored at 40°C / 75% relative humidity for 2 weeks before measurement. At all pH conditions, SDP-4 incubated for 30 min showed increased particle count compared to SDP-4 incubated for 200 min.

[0026] [Figure 4] Figure 4: Evaluation of thermal stability of buffered SDP-4 solutions. Summary graph of particle count (by Coulter method) at 1% w / w. SDP-4 solutions were reacted for 200 min and buffered with citric acid to pH 5.5. Solutions were stored for 2 weeks under the defined temperature conditions. Increasing storage temperature promoted particle formation.

[0027] [Diagram 5]Figure 5: Container tightness dramatically affects the physical stability of SDP-4. Summary of particle counts (by Coulter method) at 1.0% wt. / wt. Summary of particle counts (by Coulter method) for SDP-4 (200 min reaction) solutions stored in glass, low density polyethylene or polypropylene. No buffers or excipients were used. Solutions were stored at 40°C / 75% relative humidity for 2 weeks before measurement. Glass containers had the lowest particle counts and polypropylene containers had the highest, with low density polyethylene showing intermediate particle counts.

[0028] [Figure 6] Figure 6: Effect of buffers on the physical stability of SDP-4. Summary graph depicting microparticle counts (by Coulter method) at 1% w / w. SDP-4 solutions (240 min reaction) were buffered with glutamine, acetate or histidine at concentrations of 10 or 50 mM, pH 5.5. Solutions were stored in serum glass vials at 40°C / 75% relative humidity for 8 weeks. Glutamate and acetate buffers inhibited microparticle formation to a greater extent than histidine buffer.

[0029] [Figure 7] Figure 7: Effect of buffer and buffer strength on pH drift. Summary graph of solution pH for formulations containing 1% wt / wt. SDP-4 (240 min reaction) solutions were buffered to pH 5.5 with glutamine, acetate, or histidine buffers at 10 or 50 mM concentration. Initial pH measurements (dark, left sidebar) and remeasurements after 8 weeks (light, right sidebar) were taken at 40 °C / 75% relative humidity. Glutamate buffer was unable to maintain the pH of the SDP-4 solution, while 50 mM acetate and histidine buffers were effective in maintaining a stable pH.

[0030] [Figure 8]Figure 8: Effect of osmolarity on SDP-4 stability. Summary graph depicting the duration of solution stability until failure, defined by a count of 50 or more particulates of 10-25 μm in size. Two formulations containing 1.0% wt. / wt. SDP-4 solution (240 min reaction), 25 mM acetate buffer (pH 5.5) and mannitol as the osmotic agent were stored in glass vials at 40 °C / 75% relative humidity. The higher osmolarity solution (290 mOsm / kg) failed in 1 day, while the lower mannitol solution (180 mOsm / kg) passed up to 14 days.

[0031] [Figure 9] Figure 9: Effect of osmotic agents on the stability of SDP-4. Schematic showing the physical stability (particle count, measured by Coulter method) of 1% wt. / wt. SDP-4 (240 min reaction) formulations buffered with acetic acid (25 mM) at pH 5.5 and defined salt or sugar osmotic agents (to achieve 180 mOsm / kg). Solutions were placed in glass vials and stored at 40 °C / 75% relative humidity for 2 weeks. Mannitol and sodium chloride (NaCl) increased the particulates in the solutions, whereas MgCl2, glucose decreased particulate formation. Regardless of composition, all solutions produced more 10-25 μm particulates relative to the larger particulates measured.

[0032] [Figure 10] Figure 10: Polysorbate 80 improves the long-term stability of SDP-4. Summary graph showing the duration of solution stability until failure, defined by a particulate count greater than 50 in the 10-25 μm size range. Formulations containing 1% wt / wt. SDP-4 solution (240 min reaction), 25 mM acetate buffer (pH 5.5) and mannitol (up to 180 mOsm / kg) were stored in glass vials at 40 °C / 75% relative humidity. The addition of polysorbate 80 extended the time to failure from 1 day (control, no polysorbate 80 added) to 90 days.

[0033] [Figure 11] Figure 11: Effect of surfactant choice on physical stability of SDP-4. Summary graph depicting particulate counts at 1% wt. / wt. SDP-4 (240 min reaction) formulations were made containing 25 mM acetate buffer (pH 5.5), 38 mM magnesium chloride and 39 mM dextrose with either 0.1 wt. / wt. of polysorbate-20 or polysorbate-80. The formulations were placed in glass vials and stored under environmental conditions of 40°C / 75% relative humidity for 4 weeks and assessed for particulates by Coulter method. Formulations containing polysorbate-80 had significantly lower particulate counts than those containing polysorbate-20.

[0034] [Figure 12] FIG. 12: A questionnaire given to patients in a clinical trial using certain embodiments of the formulations disclosed herein.

[0035] [Figure 13] Figure 13: Clinical trial design flow chart for the ALPHA and BETA Phase 2 clinical trials for the treatment of dry eye.

[0036] [Figure 14A] Figure 14: The primary clinical endpoint in dry eye studies is called tear break-up time (TBUT). (A) Schematic of the standardized test procedure. [Figure 14B] (B) SDP-4 (amlisimod) increased TBUT compared to vehicle up to day 56 of the study (*p < 0.001 for SDP-4 (n=75) vs. Vehicle (n=76), Error bars = SE).

[0037] [Figure 15A] Figure 15: In subjects with dry eye subpopulation, symptoms improved significantly between treatment groups. (A) SANDE symptom scores improved the most with SDP-4 (Amlisimod) eye drops on average at day 56 (p<0.1). [Figure 15B] (B) A specific patient population that did not include patients with a SANDE score of 70 or more showed significant improvement compared to the overall population, demonstrating that SDP-4 (Amlisimod) was more effective than vehicle in this patient population (*p=0.02, Vehicle n=32 / 76, 1% SDP-4 (Amlisimod) n=38 / 76; Error bars=SE). Y-axis=change in SANDE from baseline (pts.), X-axis=days of treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Detailed Description of the Invention The present invention provides ophthalmic formulations comprising protein compositions derived from SDP. The protein compositions described herein include or can be prepared from the protein compositions described in U.S. Patent No. 9,394,355 (Lawrence et al.), which is incorporated herein by reference. The low average molecular weight fraction can also be isolated to provide compositions with enhanced anti-inflammatory activity, such as the protein compositions described in U.S. Patent Publication No. 2019 / 0169243 (Lawrence et al.), which is incorporated herein by reference.

[0039] definition The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the cited terms have the following meanings. All other terms and phrases used herein have their ordinary meanings as would be understood by one of ordinary skill in the art. Such ordinary meanings are defined in Hawley's Condensed Chemical Dictionary 14 th Edition, by R.J. Lewis, John Wiley & Sons, New York, NY, 2001.

[0040] References herein to "one embodiment," "an embodiment," and the like indicate that the described embodiment may include a particular aspect, feature, structure, portion, or characteristic, but not all embodiments necessarily include that aspect, feature, structure, portion, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment that is referenced in other parts of this specification. Furthermore, when a particular aspect, feature, structure, portion, or characteristic is described in connection with an embodiment, it is within the knowledge of one of ordinary skill in the art to affect or connect such aspect, feature, structure, portion, or characteristic to other embodiments, whether or not explicitly described.

[0041] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to an "ingredient" includes a plurality of such ingredients, such that ingredient X includes a plurality of ingredients X. Additionally, it should be noted that the claims may be drafted to exclude any element. As such, this specification is intended to serve as a predicate basis for the use of exclusive terminology, such as "solely," "only," "other than," and / or the use of recitations or "negative" limitations of claim elements in connection with any element described herein.

[0042] The term "and / or" means any one of the items, any combination of the items, or all of the items with which the term is associated. The phrases "one or more" and "at least one" are readily understood by those of ordinary skill in the art, especially when read in the context of their usage. For example, the phrase can mean 1, 2, 3, 4, 5, 6, 10, 100, or any upper limit that is approximately 10-fold, 100-fold, or 1000-fold higher than the stated lower limit.

[0043] The term "about" can refer to a variation of ±5%, ±10%, ±20%, or ±25% of the specified value. For example, "about 50" percent can, in some embodiments, carry a variation of 45 to 55 percent. For integer ranges, the term "about" can include one or two integers greater than and / or less than the recited integer at either end of the range. Unless otherwise indicated herein, the term "about" is intended to include values ​​close to the recited range, e.g., weight percent, that are equivalent in terms of the functionality of the individual components, elements, compositions, or embodiments. The term "about" can also modify the end points of the recited range, as described above in this paragraph.

[0044] As will be appreciated by those of skill in the art, all numerical values, including those expressing amounts of ingredients, properties such as molecular weight, reaction conditions, and the like, are approximate and are understood to be optionally modified in all instances by the term "about." These values ​​may vary depending on the desired properties one of skill in the art seeks to obtain utilizing the teachings of the present disclosure. It is also understood that such values ​​inherently contain variability resulting from the standard deviation found in their respective testing measurements.

[0045] As will be understood by those skilled in the art, for all purposes, particularly in terms of providing a written description, all ranges mentioned herein include all possible subranges and combinations of subranges, as well as the individual values, particularly integer values, that make up the range. A stated range (e.g., weight percent or carbon group) includes each specific value, integer value, decimal value, or identity within the range. Any stated range can be easily recognized as being fully described and allowing for breaking down the same range into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be easily broken down into a lower third, middle third, upper third, etc. Also, as will be understood by those skilled in the art, all language such as "up to," "at least," "greater than," "less than," "more than," "or more than," and the like, includes the number referenced, and such terms refer to ranges that can then be broken down into subranges, as described above. Similarly, all ratios mentioned herein include all sub-ratios that fall within the broader ratio. Thus, specific values ​​stated for radicals, substituents, and ranges are for illustration only and do not exclude other defined values ​​or other values ​​within defined ranges for radicals and substituents.

[0046] Additionally, one of ordinary skill in the art can readily recognize that where members are grouped in a common manner, such as a Markush group, the invention encompasses not only the group described as a whole, but also each member of the group individually, and all possible subgroups of the primary group. Moreover, for all purposes, the invention encompasses not only the primary group, but also the primary group excluding one or more of the group members. Thus, the invention contemplates the explicit exclusion of any one or more of the members of a recited group. Thus, provisos can be applied to any of the disclosed categories or embodiments, thereby excluding any one or more of the recited elements, species, or embodiments from such categories or embodiments, for example, for use in an express negative limitation.

[0047] The term "contacting" refers to the act of touching, coming into contact, or bringing into immediate or close proximity, including at the cellular or molecular level, for example, in a solution, in a reaction mixture, in vitro, or in vivo, to effect a physiological reaction, a chemical reaction, or a physical change.

[0048] For therapeutic applications, "effective amount" refers to an amount effective to treat a disease, disorder, and / or condition, or to produce a referenced effect. For example, an effective amount can be an amount effective to reduce the progression or severity of the condition or symptom being treated. Determination of a therapeutically effective amount is within the capabilities of one of ordinary skill in the art. The term "effective amount" is intended to include, for example, an amount of a composition described herein, or an amount of a combination of peptides described herein, effective to treat or prevent a disease or disorder, or to treat a symptom of a disease or disorder in a host. Thus, "effective amount" generally refers to an amount that provides a desired effect.

[0049] Fibroin is a protein derived from silkworm cocoons (e.g., Bombyx mori). Fibroin contains heavy chains with molecular weights of about 350-400 kDa and light chains with molecular weights of about 24-27 kDa, which are linked by disulfide bonds. The primary sequences of the heavy and light chains are known in the art. Fibroin protein chains have hydrophilic N- and C-terminal domains and alternating blocks of hydrophobic / hydrophilic amino acid sequences, allowing for mixing of steric and electrostatic interactions with surrounding molecules in solution. At low dilutions (1% or less), fibroin protein molecules are known to take the form of extended protein chains and do not immediately aggregate in solution. Fibroin protein is highly miscible with hydrating molecules such as hyaluronic acid (HA), polyethylene glycol (PEG), glycerin, and carboxymethylcellulose (CMC), demonstrating high biocompatibility, and is naturally integrated and degraded in the body by enzymatic action. Native fibroin (also referred to herein as prior art silk fibroin (PASF)) is known in the art and has been described, for example, by Daithankaret al. (Indian J. Biotechnol. 2005, 4, 115-121) and International Publication No. WO 2014 / 145002 (Kluge et.al.).

[0050] The terms "silk-derived protein" (SDP) and "fibroin-derived protein" are used interchangeably herein. These materials are prepared by the processes described herein, including heat, pressure, and high concentration heavy salt solutions. Thus, "silk-derived" and "fibroin-derived" refer to the starting material of the process of structurally modifying silk fibroin protein to arrive at a protein composition (SDP) with the structural, chemical, and physical properties described herein. SDP compositions have enhanced solubility and stability in aqueous solutions. SDP may be derived from silkworm silk (e.g., Bombyx mori), spider silk, or genetically engineered silk.

[0051] As used herein, the terms "molecular weight" and "average molecular weight" refer to the NuPAGETM Weight average molecular weight refers to the weight average molecular weight determined by standard sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis performed on 4%-12% Bis-Tris protein gels (ThermoFisher Scientific, Inc.) in combination with ImageJ software (National Institutes of Health). ImageJ is used to determine the relative amount of protein of a certain molecular weight in a sample. The software accomplishes this by converting the staining on the gel (i.e., the amount of protein) into a quantitative signal intensity. This signal is then compared to a standard (or ladder) containing biological species of known molecular weight. The amount of signal between each marker on the ladder is divided by the total signal. A cumulative sum of each protein subpopulation (also referred to herein as a fraction, and interchangeably referred to as a fragment) allows the user to determine the median molecular weight, referred to herein as the average molecular weight. In practice, electrophoretic gels are stained and then scanned into grayscale images and converted to histograms using ImageJ. The total pixel intensity within each gel lane is quantified with ImageJ (i.e., total area under the histogram) and then fractionated into populations separated by the protein molecular weight standards stained on the gel. The percentage of total protein contained within these molecular weights is calculated by dividing the histogram pixel area between the two molecular weight standards by the total histogram area of ​​the protein. Analysis by other methods may yield different values ​​that account for specific peptides not accounted for by the SDS-PAGE method. For example, average molecular weights can be analyzed using HPLC, which typically yields values ​​approximately 10-30% lower than those determined by SDS-PAGE (the difference increases as the molecular weight decreases).

[0052] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present disclosure provides formulations comprising: (a) a fibroin-derived protein composition, the primary amino acid sequence of the fibroin-derived protein composition differs from that of native fibroin by at least 4% in terms of the absolute value of the sum of the differences in amino acid content of serine, glycine, and alanine; and the fibroin-derived protein composition has reduced or eliminated cysteine ​​disulfide bonds between the fibroin heavy protein chain and the fibroin light chain. The protein composition has a serine content that is reduced by 25% or more compared to native fibroin, where the serine content is at least about 5%; and the average molecular weight of the fibroin-derived protein composition is 15-35 kDa; and comprises (b) a buffering agent, (c) polysorbate-80, and (d) one or more osmotic agents such that the mOsm is between 170 mOsm / kg and about 300 mOsm / kg; where the formulation has a pH of 4.5-6.0 and has a particulate count of 50 / mL or less for particulates having a diameter of 10 micrometers or greater after a storage period of 12 weeks or more at 4°C to 40°C.

[0053] In some embodiments, the protein composition comprises more than 46.5% glycine amino acids, or the protein composition comprises more than 30.5% alanine amino acids or more than 31.5% alanine amino acids. In other embodiments, the protein composition has a serine content that is reduced by more than 40% compared to the native fibroin protein, such that the protein composition comprises less than 8% serine amino acids. In additional embodiments, more than 50% of the protein chains of the protein composition have a molecular weight in the range of 10 kDa to 40 kDa.

[0054] In further embodiments, the primary amino acid sequence of the fibroin derived protein composition differs from native fibroin by at least 6% with respect to the combined difference in serine, glycine, and alanine content; and the average molecular weight of the fibroin derived protein is between 12 kDa and 30 kDa. In various other embodiments, the fibroin derived protein composition is silk-derived protein-4 (SDP-4) having an average molecular weight of between about 15 kDa and about 35 kDa, and the pH of the formulation is between about 5.0 and about 6.0. In other embodiments, the pH is between 5.2 and 5.8.

[0055] In various embodiments, the osmolality of the formulation is about 170 mOsm / kg to about 300 mOsm / kg, in some embodiments, the osmolality is about 160 mOsm / kg to about 200 mOsm / kg, about 175 mOsm / kg to about 180 mOsm / kg, about 180 mOsm / kg to about 200 mOsm / kg, about 200 mOsm / kg to about 250 mOsm / kg, or about 250 mOsm / kg to about 300 mOsm / kg.

[0056] Weight percent expressions are to be interpreted as % wt. / wt in this disclosure. In various embodiments, the weight percent of SDP-4 in the formulation is about 0.01% to about 15%. In additional embodiments, the weight percent of SDP-4 is about 0.1% to about 5%, or about 0.1%, about 1%, or about 3%. In some embodiments, the buffering agent comprises histidine, acetate, glutamate, or a combination thereof. In still other embodiments, the formulation has a buffer concentration of about 10 millimolar to about 50 millimolar, or about 20 millimolar to about 40 millimolar. In other embodiments, the concentration of each of the one or more osmotic agents in the formulation is about 30 millimolar to about 40 millimolar, or about 35 millimolar. In other embodiments, the buffering agent comprises about 0.1% to about 1.0% by weight sodium acetate and about 0.01% to about 0.1% by weight acetic acid. In another embodiment, the buffer is composed of about 0.5% to about 2.0% sodium acetate by weight and about 0.05% to about 1.0% acetic acid by weight.

[0057] In other embodiments, the osmotic reagent comprises a monosaccharide, an inorganic salt, or a combination thereof. In additional embodiments, the osmotic reagent comprises mannitol, dextrose, sodium chloride, magnesium chloride, or a combination thereof. In other embodiments, the osmotic reagent comprises about 0.1% to about 2% by weight of glucose and about 0.1% to about 2% by weight of magnesium chloride. In yet other embodiments, the osmotic reagent comprises about 0.01% to about 2% by weight of dextrose and about 0.01% to about 2% by weight of magnesium chloride. In further embodiments, the weight percent of polysorbate-80 is about 0.02% to about 2%. In other embodiments, the weight percent of polysorbate-80 is about 0.01% to about 2%. In additional embodiments, the formulation is stored in a container comprising glass or polyethylene. In various embodiments, the container is a type I borosilicate glass. In additional embodiments, the container can be a low density polyethylene container. The formulation has been shown to be stable for greater than six months in low density polyethylene containers.

[0058] In other embodiments, the storage period or shelf life is about 4 months to about 8 months, about 8 months to about 12 months, about 1 year to about 2 years, or more than 2 years from the date of manufacture. In various embodiments, the microparticle count after storage is about 200 / mL, about 150 / mL, about 100 / mL, about 75 / mL, about 45 / mL, about 35 / mL, about 25 / mL, about 20 / mL, about 15 / mL, about 10 / mL, about 5 / mL, or about 1 / mL. In yet other embodiments, the storage temperature is about 10°C to about 30°C, or about 15°C to about 25°C.

[0059] The disclosure also provides an aqueous formulation comprising about 0.1% to about 3% by weight, wherein the primary amino acid sequence of SDP-4 differs from native fibroin by at least 6% in terms of the absolute value of the total difference in amino acid content of serine, glycine, and alanine, SDP-4 has reduced or eliminated cysteine ​​disulfide bonds between the fibroin heavy and fibroin light protein chains, SDP-4 is composed of more than 46% glycine amino acids and more than 30% alanine amino acids, and the SDP-4 has more than 46% of the amino acids of native fibroin. The SDP-4 has a serine content that is reduced by 40% or more compared to native fibroin protein such that it contains less than 8% serine amino acids; and the average molecular weight of the SDP-4 is about 15 kDa to about 35 kDa; and comprises polysorbate-80, about 10 mM to about 50 mM acetate buffer, an osmotic agent; wherein the formulation has a pH value of 5.2 to 5.8; and has an osmotic agent; wherein the formulation is characterized in that the SFP-4 is 5.2 to 5.8, has an osmolality of 175 mOsm / kg to 185 mOsm / kg, and has a particulate count of 50 mL or less after a storage period of more than 12 weeks at 4° C. to 40° C. for particulates having a diameter of 10 micrometers or greater.

[0060] In a preferred embodiment, the formulation may consist essentially of a fibroin-derived protein composition, the primary amino acid sequence of which differs from native fibroin by at least 4% in terms of the absolute value of the sum of the differences in amino acid content of serine, glycine, and alanine, and the cysteine ​​disulfide bonds between the fibroin heavy protein chain and the fibroin light chain of the fibroin-derived protein are reduced or eliminated, the protein composition having a serine content reduced by 25% or more compared to native fibroin, the serine content being at least about 5%, the fibroin-derived protein composition having an average molecular weight of less than 35 kDa and greater than 15 kDa, and comprising a buffer, polysorbate-80, and one or more osmotic agents, wherein the formulation has a pH of 4.5-6.0, and is characterized in that for microparticles having a diameter of 10 μm or greater, the formulation has a microparticle count of 50 / mL or less after a storage period of more than 12 weeks at 4° C.-40° C.

[0061] In another preferred embodiment, the formulation can consist essentially of about 0.1% to about 3% by weight SDP-4, wherein the primary amino acid sequence of the fibroin-derived protein differs from that of native fibroin by at least 6% in terms of the absolute value of the sum of the differences in amino acid content of serine, glycine, and alanine, the cysteine ​​disulfide bonds between the fibroin heavy protein chain and the fibroin light protein chain of the fibroin-derived protein are reduced or eliminated, the fibroin-derived protein contains more than 46% glycine amino acids and more than 30% alanine amino acids, and the fibroin-derived protein contains less than 8% serine amino acids. and has a serine content that is reduced by 40% or more compared to native fibroin protein so as to include amino acids, an average molecular weight of SDP-4 is about 15 kDa to about 35 kDa, and comprises polysorbate-80, about 10 mM to about 50 mM acetate buffer, an osmotic agent, the formulation being characterized in that it has a pH of 5.2 to 5.8, an osmolality of 175 mOsm / kg to 185 mOsm / kg, and a particulate count of 50 mL or less after a storage period of more than 12 weeks at 4° C. to 40° C. for particulates having a diameter of 10 micrometers or greater.

[0062] In various embodiments, the acetate buffer is comprised of about 0.2% to about 0.3% sodium acetate and about 0.01% to about 0.03% acetic acid by weight. In some embodiments, the osmotic agent comprises about 0.6% to about 0.9% glucose and about 0.6% to about 0.9% magnesium chloride by weight. In additional embodiments, the weight percent of polysorbate-80 is about 0.05% to about 0.1%.

[0063] In various embodiments, the formulation is as prepared herein, with the fibroin derived protein (e.g., SDP-4) present at a final concentration of about 0.1% w / w, sodium acetate present at a final concentration of about 0.25% w / w, glacial acetic acid present at a final concentration of about 0.01% w / w, magnesium chloride present at a final concentration of about 0.8% w / w, dextrose at a final concentration of about 0.8% w / w, and polysorbate-80 at a final concentration of about 0.05% w / w.

[0064] In various embodiments, the formulation may be a fibroin derived protein (e.g., SDP-4 as prepared herein present at a final concentration of about 1% w / w), sodium acetate present at a final concentration of about 0.25% w / w, glacial acetic acid present at a final concentration of about 0.01% w / w, magnesium chloride present at a final concentration of about 0.75% w / w, dextrose present at a final concentration of about 0.75% w / w, and polysorbate-80 present at a final concentration of about 0.05% w / w.

[0065] In some embodiments, the formulation may include a fibroin derived protein (e.g., SDP-4 prepared herein present at a final concentration of about 3% w / w), sodium acetate present at a final concentration of about 0.25% w / w, glacial acetic acid at a final concentration of about 0.01% w / w, magnesium chloride present at a final concentration of about 0.65% w / w, dextrose present at a final concentration of about 0.65% w / w, and polysorbate-80 present at a final concentration of about 0.05% w / w.

[0066] Further, the present disclosure provides a method of treating an ophthalmic disease, comprising administering to a subject having the ophthalmic disease an effective amount of the formulation disclosed above, thereby treating the ophthalmic disease. In some embodiments, the ophthalmic disease is dry eye syndrome.

[0067] The formulations described herein provide effective treatment and / or symptom relief for eye-related conditions. These results are surprising, at least in part, because common techniques discourage those of skill in the art from selecting the specific combination of ingredients used in our formulations. For example, Wang et al., Dual Effects of Tween 80 on Protein Stability., Int J Pharm.2008 Jan 22;347(1-2):31-8, directed to studying the effect of Tween-80 on the stability and aggregation of the model protein IL-2, discloses that "the addition of 0.1% Tween 80 significantly increased the rate of IL-2 mutein aggregation during storage" (Wang, Abstract, p. 31). However, the inventors have found that the use of polysorbates (e.g., polysorbate 80) as surfactants in the formulations significantly inhibits protein aggregation in solution.

[0068] Moreover, our formulations unexpectedly exhibit properties that are inconsistent with the prevailing art. Katakam et al., Effects of Surfactants on the Physical Stability of Recombinant Human Growth Hormone. J Pharm Sci. 1995 Jun;84(6):713-6 (Effects of Surfactants on the Physical Stability of Recombinant Human Growth Hormone) concerns the effect of certain surfactants (e.g., BRIJ 35, TWEEN-80, Pluronic F68) on the physical stability of human growth hormone following exposure to the air / water interface and non-isothermal stress. Katakam discloses that TWEEN-80 (i.e., polysorbate 80) did not protect hGH from heat stress: "Surfactants at concentrations that stabilize hGH with respect to interfacial denaturation (by agitation) did not provide any protection against heat stress." (Katakam, page 716, 2nd full para.). In contrast, the present inventors have found that the use of TWEEN-80 (polysorbate 80) protects proteins in solution from heat stress.

[0069] Kreilgaard et al., Effect of Tween 20 on Freeze-Thawing- and Agitation-induced Aggregation of Recombinant Human Factor XIII., J Pharm Sci. 1998 Dec;87(12):1597-603, was directed to investigating the effect of polysorbate 20 (i.e., Tween 20) on freeze-thaw-induced aggregation of recombinant human factor XIII (rFXIII). Kreilgaard discloses that "these observations suggest that Tween 20 stabilizes rFXIII [protein] primarily by competing with stress-induced soluble aggregates for the interface and inhibiting their subsequent transition to insoluble aggregates" (Kreilgaard, page 1602, last full para.). Furthermore, Bam et al., Tween Protects Recombinant Human Growth Hormone against Agitation-Induced Damage via Hydrophobic Interactions., J Pharm Sci. 1998 Dec;87(12):1554-9, disclose that "in the absence of detergent, recombinant human growth hormone rapidly forms insoluble aggregates during agitation." The non-ionic detergent TWEEN-20 effectively inhibits this aggregation when present at a detergent:protein molar ratio >4 (Bam, Abstract). These studies present the opposite of what we found: the use of polysorbate 20 (TWEEN-20) actually destabilizes proteins in solution, leading to increased aggregation and the formation of insoluble particulates.

[0070] Additionally, common techniques teach the use of osmolytes / polyols such as glycerol to prevent protein aggregation in solution. For example, Vagenende et al., Mechanisms of Protein Stabilization and Prevention of Protein Aggregation by Glycerol., Biochemistry 2009 Nov 24;48(46):11084-96, state that "glycerol inhibits protein unfolding and prevents protein aggregation by stabilizing aggregation-prone partially unfolded intermediates through preferential interactions with hydrophobic surface regions that favor the orientation of glycerol's amphiphilic interface" (Vagenende, page 11094, 4 thSimilarly, Feng et al., Effects of glycerol on the compaction and stability of the wild type and mutated rabbit muscle creatine kinase., Proteins 2008 May 1;71(2):844-54 disclose that in the presence of glycerol in the refolding buffer, "aggregation of both proteins behaved similarly: it decreased with increasing glycerol concentration and was completely inhibited at 30% glycerol" (Feng, page 850, first paragraph). (Prieve et al., Glycerol decreases the Volume and Compressibility of Protein Interior., Biochemistry 1996, 35, 2061-2066: "We suggest that glycerol induces the release of so-called 'lubricating' water, which maintains conformational flexibility by moving adjacent segments of the polypeptide chain apart (thus enhancing protein stability)" (Prieve, Abstract); Gekko et al., Mechanism of Protein Stabilization: Preferential Hydration in Glycerol-Water Mixtures., Biochemistry 1981, 20, 4667-4676: "Present measurements of preferential interactions of proteins with solvent components in the water-glycerol solvent system indicate that of six proteins examined, all are preferentially hydrated in this solvent system...it therefore seems reasonable to generalize this situation to other proteins."Moreover, it has long been known empirically that protein conformation is stabilized by the presence of glycerol (Gekko, page 4674, 2nd full para.); Sedgwick et al., Protein Phase Behavior and Crystallization: Effect of Glycerol., J. Chem. Phys. 2007 Sep 28;127(12):125102, "We found that at a constant protein concentration, increasing amounts of salt were required to crystallize the protein, and that increasing glycerol concentrations progressively increased the time required for crystallization" (Sedgwick, page 6, 3rd full para)). Surprisingly, the inventors have found that the use of glycerol in the formulation significantly promoted protein aggregation and the formation of insoluble microparticles.

[0071] Additionally, Chen et al., Influence of Histidine on the Stability and Physical Properties of a Fully Human Antibody in Aqueous and Solid Forms., Pharm Res. 2003 Dec;20(12): 1952-60 disclose the utility of using histidine to prevent protein aggregation: "Increasing the histidine concentration in the bulk solution was found to inhibit the increase in high molecular weight (HMW) species and aggregates upon lyophilization and storage. Furthermore, bulk histidine improved the solution stability of the antibody under freezing and heat stress conditions, as evidenced by lower levels of aggregates" (Chen, Abstract). Furthermore, Shiraki et al., Amino Acid Esters Prevent Thermal Inactivation and Aggregation of Lysozyme, Biotechnol Prog. 2005 21: 640-643 teaches the advantageous use of amino acid esters in preventing heat inactivation of proteins: "Amino acid esters (AAEs) prevent heat-induced aggregation and inactivation of hen egg lysozyme". Lysozyme was completely inactivated (less than 1% of original activity) by heat treatment at 98°C for 30 minutes in a solution containing 0.2 mg / mL lysozyme in 50 mM Na-phosphate buffer (pH 6.5)" (Shiraki, abstract). In contrast to these studies, the present inventors found that low concentrations of amino acid esters (arginine) did not prevent protein aggregation, and the use of high concentrations of amino acid esters led to gelation of the formulation. Also, histidine increased the stability of proteins in solution, but was found to be unsuitable for ophthalmic formulations due to the irritating properties of histidine to the eye.

[0072] Regarding the use of buffers and ions with formulations, the general art teaches the benefits of using calcium ions to stabilize proteins in solution. For example, Saboury et al., Effects of calcium binding on the structure and stability of human growth hormone., Int J Biol Macromol. 2005 Sep 28;36(5):305-9 discloses that "calcium ion binding increases the thermal stability of proteins by increasing α-helical content and decreasing both β and random coil structures" (Sarboury, Abstract). Furthermore, Pikal-Cleland et al., Effect of glycine on pH changes and protein stability during freeze-thawing in phosphate buffer systems., J Pharm Sci. 2002 Sep;91(9):1969-79 teach the advantage of using glycine to minimize the formation of discrete pH microenvironments during solution freezing that are the basis of protein instability: "The presence of high concentrations (>100 mM) of glycine in sodium phosphate buffers resulted in more complete crystallization of the disodium salt as indicated by freezing pH values ​​closer to the equilibrium value (pH 3.6)" (Pikal-Cleland, Abstract). However, the inventors found that the use of calcium ions negatively affected the solubility of proteins in solution, while the use of glycine had no appreciable effect on protein stability.

[0073] Given the teachings of the art in general, one of ordinary skill in the art would not be motivated to pursue a formulation containing the combination of ingredients selected by applicant, and one of ordinary skill in the art would not be able to manufacture the formulation and related functions with a reasonable expectation of success that the formulation would be effective in treating ocular-related conditions, particularly dry eye disease.

[0074] Preparation of SDP Composition Protein compositions for use in ophthalmic formulations can be prepared as described in U.S. Patent No. 9,394,355 (Lawrence et al.) and U.S. Patent Publication No. 2019 / 0169243 (Lawrence et al.). The SDP can be derived from Bombyx mori silkworm fibroin or other fibroins or other silk proteins of the Bombyx genus.

[0075] The SDP material can be prepared by the following steps. 1. Silk cocoons are prepared by removing any pupal material and pre-washing in warm water. 2. By washing the cocoons with alkaline pH water at high temperatures, usually above 95°C, the fibroin protein fibrils can be extracted from the gummy sericin protein. 3. The extracted fibroin fibers are dried and then dissolved in a solvent system that neutralizes the hydrogen bonds between the β-sheets. A solution of 20% w / v silk fibroin protein in 54% LiBr in water is effective for this neutralization step. 4. Treat the fibroin protein dissolved in LiBr solution in an autoclave environment (~121 °C [~250 °F], pressure ~15-17 PSI, temperature ~30 min). 5. The heat-treated fibroin protein and LiBr solution are dialyzed to remove the lithium and bromide ions in solution. At this point, the material has been chemically transformed into SDP. 6. The dialyzed SDP is filtered to remove non-soluble aggregates and contaminating bioburden.

[0076] The SDP solution is produced using a process that is distinctly different from that used to produce current silk fibroin solutions. Of note, autoclaving the silk fibroin protein in LiBr solution (not after removing the LiBr) initiates a chemical transition that produces a stabilized SDP material. The fibroin protein dissolves in the LiBr solution, neutralizing the hydrogen bonds and electrostatic interactions of the solubilized native fibroin protein. Under these conditions, the protein cannot assume a specific secondary structure in solution. As a result, the thermodynamic energy required to hydrolyze the covalent bonds within the fibroin protein chains is lowest, facilitating hydrolytic cleavage and conformational changes following the formation of dehydro-alanine structures.

[0077] SDP preparation conditions include temperature set at 121°C for 30 minutes at 15-17 PSI in an autoclave. However, in various embodiments, processing conditions may be altered to stabilize the SDP material to various degrees. Additional protein solubilizers may be used in the process, including other or additional halide salts such as calcium chloride and sodium thiocyanate, organic agents such as urea, guanidine hydrochloride and 1,1,1,3,3,3-hexafluoroisopropanol, additional strong ionic liquid solution additives such as calcium nitrate and 1-butyl-3-methylimidazolium chloride, or combinations thereof.

[0078] SDP Composition The SDP compositions described herein are derived from silk fibroin and can have enhanced solubility and stability in aqueous solutions. The compositions can be used to treat and reduce inflammation. In one embodiment, the SDP and / or fractions thereof have a primary amino acid sequence that differs from native fibroin by at least 4% (through the sum of the absolute values ​​of the differences) with respect to the combined amino acid content of serine, glycine, and alanine. In some embodiments, a plurality of protein fragments of the SDP can terminate with amide (-C(=O)NH2) groups. The SDP can have a serine content that is reduced by 40% or more compared to native fibroin, where the serine content is at least about 100%. Cysteine ​​disulfide bonds between the silk fibroin and the fibroin light protein chains can be reduced or eliminated. In certain embodiments, at least 75% of the protein fragments have a molecular weight of less than about 60 kDa. The composition can include less than 8.5% serine amino acid residues. In some embodiments, the average molecular weight of the SDP is less than 55 kDa. The SDP composition has enhanced stability in aqueous solutions.

[0079] In some embodiments, the SDP protein composition is prepared by a process that includes heating an aqueous fibroin solution at elevated pressure. The aqueous fibroin solution is 5%. The solution includes lithium bromide at a concentration of at least 8 M. The aqueous fibroin solution is heated to at least about 105° C. (221° F.) under a pressure of at least about 10 PSI for at least about 20 minutes to provide a protein composition. As a result of these treatment conditions, the polypeptides of the protein composition contain less than 8.5% serine amino acid residues and a plurality of protein fragments terminated in amide (C(═O)NH2) groups.

[0080] In some embodiments, the SDP composition is prepared by a process comprising heating an aqueous fibroin solution at high pressure, the aqueous fibroin solution comprising lithium bromide at a concentration of 9-10 M, and heating the aqueous fibroin solution to a temperature ranging from about 115° C. (239° F.) to about 125° C. (257° F.) under a pressure of about 15 PSI to about 20 PSI for at least about 20 minutes; providing a protein composition. The protein composition can contain less than 6.5% serine amino acid residues.

[0081] In some aspects, the preparation method can use lithium bromide having a concentration between about 8.0 M and about 11 M. In some embodiments, the concentration of lithium bromide is about 9 M to about 10 M, or about 9.5 M to about 10 M.

[0082] In some embodiments, the aqueous fibroin solution containing lithium bromide is heated to at least about 107°C (225°F), at least about 110°C (230°F), at least about 113°C (235°F), at least about 115°C (239°F), or at least about 120°C (248°F).

[0083] In some embodiments, the aqueous fibroin solution containing lithium bromide is heated under a pressure of at least about 12 PSI, at least about 14 PSI, at least about 15 PSI, or at least about 16 PSI, up to about 18 PSI, or up to about 20 PSI.

[0084] In some embodiments, the aqueous fibroin solution containing lithium bromide is heated for at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, or at least about 1 hour, up to several hours (e.g., 12-24 hours).

[0085] The SDP composition is chemically distinct from native silk fibroin protein, resulting from the preparation process resulting in altered amino acid content and the formation of terminal amide groups. The resulting SDP has improved solubility and stability in aqueous solutions. The SDP can be used in methods for forming, for example, ophthalmic formulations using the protein compositions described herein, for example, in aqueous solutions of the protein compositions. The solutions can include about 0.01% to about 35% w / v SDP. The solutions can be about 65% to about 99.9% w / v water.

[0086] In some embodiments, the SDP is prepared using a process that induces hydrolysis, amino acid degradation, or a combination thereof, of the fibroin protein such that the average molecular weight of the protein is reduced from about 100-200 kDa for silk fibroin produced using prior art methods to about 35-90 kDa, or about 40-50 kDa for the SDP materials described herein. The resulting polypeptide can be a random assortment of peptides of various molecular weights averaged into the ranges described herein.

[0087] Additionally, the amino acid chemistry can be altered by reducing the cysteine ​​content to levels undetectable by standard assay procedures. For example, the serine content can be reduced by 50% or more from levels found in native fibroin, which can result in a 5% increase in overall alanine and glycine content (relative amino acid content) as determined by standard assay procedures. The SDP material can have a serine content of less than about 8% relative amino acid content, or a serine amino acid content of less than about 6% relative amino acid content. The SDP material can have a glycine content of greater than about 46.5%, and / or an alanine content of greater than about 30%, or an alanine content of greater than about 30.5%. The SDP material can have no detectable cysteine ​​content, for example, as determined by HPLC analysis of hydrolyzed polypeptides of the protein composition. The SDP material can form 90% less, 95% less, or 98% less beta-sheet secondary protein structure compared to native silk fibroin protein, for example, as determined by FTIR analysis.

[0088] The SDP composition has enhanced stability in aqueous solution, where: the primary amino acid sequence of the SDP composition differs from native fibroin by at least 4% in terms of the difference in the sum (absolute value) of serine, glycine, and alanine content (SDP vs. PASF); the cysteine ​​disulfide bond between the fibroin heavy protein chain and the fibroin light chain is reduced or eliminated, and the composition has a serine content that is reduced by more than 25% compared to native fibroin protein. The average molecular weight of the SDP composition can be less than 60 kDa, greater than about 35 kDa, or greater than about 40 kDa, as determined by the MWCO of the dialysis membrane and SDS-PAGE analysis.

[0089] In some embodiments, the SDP composition has a primary amino acid sequence that differs from native fibroin by at least 6% in terms of the combined difference in serine, glycine, and alanine content; the cysteine ​​disulfide bonds between the fibroin heavy protein chain and the fibroin light protein chain are reduced or eliminated; and the composition has a serine content that is reduced by 40% or more compared to native fibroin protein. The average molecular weight of the SDP composition can be less than about 55 kDa and greater than about 35 kDa, as determined by the MWCO of the dialysis membrane and SDS-PAGE analysis.

[0090] In some embodiments, the SDP composition has a primary amino acid sequence that is altered from native silk fibroin; the cysteine ​​disulfide bond between the fibroin heavy protein chain and the fibroin light chain is reduced or eliminated; the average molecular weight of the SDP composition is less than about 60 kDa and greater than about 35 kDa; and a 5% w / w aqueous solution of the SDP composition maintains an optical absorbance at 550 nm of less than 0.25 for at least 2 hours after being sonicated for 5 seconds.

[0091] In some embodiments, the SDP composition has enhanced stability in aqueous solution, where: the primary amino acid sequence of the SDP composition is modified from native silk fibroin to differ from native fibroin by at least 5% in terms of the difference in the sum of serine, glycine, and alanine content (absolute value). In some embodiments, the difference is at least 6%, 8%, 10%, 12%, or 14% compared to native fibroin; the cysteine ​​disulfide bond between the fibroin heavy protein chain and the fibroin light protein chain is reduced or eliminated; the average molecular weight of the SDP composition is less than about 60 kDa, greater than about 35 kDa; and the SDP composition maintains an absorbance at 550 nm of less than 0.2 for at least 2 hours after 5 seconds of sonication.

[0092] The SDP compositions can be isolated and / or purified, for example, by dialysis and / or filtration, as dry powders or films, or alternatively, as stable aqueous solutions, which can be modified for use as therapeutic formulations, such as the ophthalmic formulations described herein.

[0093] In various embodiments, the amino acid composition of the SDP can differ from the amino acid composition of native fibroin by at least 4%, at least 4.5%, at least 5%, or at least 5.5%, or at least 6% in terms of the combined serine, glycine, and alanine content.

[0094] In some embodiments, the SDP compositions described herein have a serine content that is reduced by more than 25%, more than 30%, more than 35%, more than 40%, or more than 45% compared to the serine content of native fibroin protein.

[0095] The average molecular weight of the SDP composition can be less than about 80 kDa, less than about 70 kDa, less than about 60 kDa, or less than about 55 kDa, or the composition has an average molecular weight of about 50-60 kDa, or about 51-55 kDa. In various embodiments, the average molecular weight of the SDP composition can be greater than 35 kDa, greater than about 40 kDa, or greater than about 50 kDa. Thus, the (weight average) average molecular weight of the SDP composition can be about 36 kDa to about 80 kDa, about 36 kDa to about 65 kDa, about 36 kDa to about 60 kDa, or about 40 kDa to about 55 kDa. In various embodiments, the average molecular weight of the SDP composition is from about 45 kDa to about 65 kDa, from about 45 kDa to about 60 kDa, from about 50 kDa to about 65 kDa, or from about 50 kDa to about 60 kDa.

[0096] The SDP composition can be soluble in water at 40% w / w without producing any precipitation observable by ocular examination.

[0097] In some embodiments, the SDP composition comprises less than 8% serine amino acid residues. In some embodiments, the protein composition comprises less than 7.5% serine amino acid residues, less than 7% serine amino acid residues, less than 6.5% serine amino acid residues, or less than 6% serine amino acid residues. The serine content of the peptide composition is generally at least about 4%, or at least about 5%, or about 4-5%.

[0098] In some embodiments, the SDP composition comprises greater than 46.5% glycine amino acids relative to the total amino acid content of the protein composition. In some aspects, the protein composition comprises greater than 47% glycine amino acids, greater than 47.5% glycine amino acids, or greater than 48% glycine amino acids.

[0099] In some embodiments, the SDP composition comprises greater than 30% alanine amino acids relative to the total amino acid content of the protein composition, hi some aspects, the protein composition comprises greater than 30.5% alanine, greater than 31% alanine, or greater than 31.5% alanine.

[0100] In some embodiments, the SDP composition can be completely redissolved after drying to a thin film. In various embodiments, the protein composition can lack beta sheet protein structure in aqueous solution. The protein composition can maintain an optical absorbance in aqueous solution of less than 0.25 at 550 nm after sonication for at least 5 seconds.

[0101] In some embodiments, the SDP protein composition can be combined with water. In some embodiments, the protein composition can be completely dissolved in water at a concentration of 10% w / w, or even higher concentrations such as 15% w / w, 20% w / w, 25% w / w, 30% w / w, 35% w / w, or 40% w / w. In some embodiments, the protein composition can be isolated and purified, for example, by dialysis, filtration, or a combination thereof.

[0102] In various embodiments, the SDP compositions can, for example, improve the spreading of an aqueous solution comprising a protein composition and an ophthalmic formulation component as compared to the spreading of a corresponding composition without the protein composition, and this enhanced spreading can result in a two-fold or greater, or three-fold or greater increase in the surface area of ​​the aqueous solution.

[0103] In various embodiments, the SDP composition does not form gels in water at concentrations up to 20% w / v, up to 30% w / v, or up to 40% w / v. In some embodiments, the SDP composition can have glycine-alanine-glycine-alanine (GAGA) (SEQ ID NO:1) segments of amino acids that make up at least about 47.5% of the amino acids of the SDP composition. In some cases, the SDP composition can also have GAGA (SEQ ID NO:1) segments of amino acids that make up at least about 48%, at least about 48.5%, at least about 49%, at least about 49.5%, or at least about 50% of the amino acids of the protein composition.

[0104] In various embodiments, the SDP composition can have glycine-alanine (GA) segments of amino acids that make up at least about 59% of the amino acids of the SDP composition. In some cases, the SDP composition can also have GA segments of amino acids that make up at least about 59.5%, at least about 60%, at least about 60.5%, at least about 61%, or at least about 61.5% of the amino acids of the protein composition. In typical embodiments, the fibroin is separated from the sericin. In various embodiments, the SDP composition redissolves as a thin film after drying, a property not seen in native fibroin.

[0105] In a specific embodiment, the present invention provides an SDP composition prepared by a process comprising heating an aqueous fibroin solution at high pressure, the aqueous fibroin solution comprising a lithium bromide concentration of 9-10 M, and the aqueous fibroin solution is heated to a temperature in the range of about 115° C. (239° F.) to about 125° C. (257° F.) for at least about 30 minutes under a pressure of about 15 PSI to about 20 PSI; wherein the protein composition comprises less than 6.5% serine amino acid residues; comprises 5% serine amino acid residues; and wherein the protein composition has an aqueous viscosity of less than 10 cP as a 15% w / w solution in water.

[0106] Stability Assessment The stability of a protein solution can be assessed in many different ways. One suitable assessment is the Lawrence Stability Test (US Pat. No. 9,394,355 (Lawrence et al.,)). Another suitable assessment is to subject a protein solution to sonication followed by absorbance analysis to confirm continued optical clarity (and lack of aggregation, beta-sheet formation, and / or gelation). Standard sonication or alternative sonication (sound frequency >20 kHz) can be used to test the stability of SDP solutions. Solutions of SDP are stable after being subjected to sonication. The SDP composition maintains an optical absorbance at 550 nm of less than 0.25 for at least 2 hours after 5 seconds of sonication. For example, a 5% w / w solution of the protein composition maintains an optical absorbance at 550 nm of less than 0.1 after 5 seconds of sonication at ~20 kHz, the standard conditions used for sonication described herein. In various embodiments, the aqueous SDP composition solution does not gel when sonicated at concentrations up to 10% w / w, hi further embodiments, the aqueous SDP composition solution does not gel when sonicated at concentrations up to 15% w / w, up to 20% w / w, up to 25% w / w, up to 30% w / w, up to 35% w / w, or up to 40% w / w.

[0107] Low-viscosity SDP has a lower viscosity than native silk fibroin (PASF) as a result of its preparation process and the resulting change in the chemical structure of the peptide chain. As a 5% w / w solution in water (25.6°C), the viscosity of native silk fibroin is about 5.8 cP, while under the same conditions, SDP has a viscosity of about 1.8 cP and SDP-4 has a viscosity of about 2.7 cP (e.g., 2.6-2.8 cP). SDP maintains a low viscosity compared to PASF even at high concentrations. The SDP composition can have an aqueous viscosity of less than 5 cP, or less than 4 cP, as a 10% w / w solution in water. In various embodiments, the SDP remains in solution up to a viscosity of at least 9.8 cP. The SDP can also have an aqueous viscosity of less than 10 cP as a 15% w / w solution in water. The SDP can also have an aqueous viscosity of less than 10 cP as a 24% w / w solution in water.

[0108] The process described herein involves the step of: TM The present invention provides a protein composition in which fibroin light chain proteins are not identifiable after processing, even when run using standard sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis methods performed on 4%-12% Bis-Tris protein gels (ThermoFisher Scientific, Inc.). Furthermore, while silk fibroin solutions produced using prior art methods form significant amounts of β-sheet secondary structure, the resulting SDP material is capable of post-processing that results in minimal to no β-sheet protein secondary structure. In one embodiment, the SDP material can be prepared by processing silk fibroin fibers under autoclave or autoclave-like conditions (i.e., about 120° C. and 14-18 PSI) in the presence of a 40-60% w / v lithium bromide (LiBr) solution.

[0109] SNP composition Silk Technologies, Ltd. has developed a silk-derived protein (SDP) product that can be easily incorporated into ophthalmic product formulations to reduce inflammation and enhance the wound healing process. The SDP can be separated into smaller protein fractions or subpopulations based on molecular weight to enhance anti-inflammatory and wound healing activity. The SDP protein subpopulations, also called fractions or fragments, can be separated by any suitable and effective method, for example, size exclusion chromatography or membrane dialysis. For example, the fractions can be separated into two to four different groups based on decreasing average molecular weight. Example 6 describes one method for preparing four different fractions with the same overall amino acid content and terminal amide content, but different average molecular weights. It has been surprisingly discovered that the different cellular uptake of the different fractions results in different biological properties, for example, properties for reducing inflammation in the body and in various tissues.

[0110] The low average molecular weight fraction of SDP reduces inflammation and treats dry eye. Also described are compositions for treating ocular conditions, including, but not limited to, dry eye disease and / or injuries, including corneal wounds. Therapeutic methods can include administration of SDP or a formulation comprising a low molecular weight SDP subpopulation (SDP-4). In certain embodiments, the present invention provides a method for treating a disease state and / or wound comprising administering a composition comprising a low molecular weight SDP (e.g., SDP-4) to a subject in need thereof.

[0111] SDP-4 is a subpopulation of SDP proteins having a primary amino acid sequence (via sum of absolute differences) that differs from native fibroin by at least 4% with respect to the combined amino acid content of serine, glycine, and alanine. The plurality of protein fragments can terminate with amide (-C(=O)NH2) groups. SDP-4 compositions have a serine content that is reduced by 40% or more compared to native fibroin, where the serine content is at least about 5%. Cysteine ​​disulfide bonds between the fibroin heavy and fibroin light protein chains of fibroin may be reduced or eliminated. In some embodiments, at least 75% of the protein fragments have a molecular weight of less than about 100 kDa. Such compositions reduce inflammation, promote cell migration and / or proliferation in tissues, treat disease conditions, and / or promote wound closure. SDP compositions have enhanced solubility and stability in aqueous solutions.

[0112] The SDP composition fraction can have an average molecular weight between about 15 kDa and 60 kDa. In one embodiment, a low molecular weight fraction is isolated having an average molecular weight between about 15 and 35 kDa, and this fraction is designated SDP-4.

[0113] In some embodiments, at least 60 percent of the protein fragments have a molecular weight of less than about 60 kDa, or less than about 55 kDa, to promote cell migration and proliferation in tissue to close the wound, hi another embodiment, at least 90% of the protein fragments have a molecular weight of less than about 100 kDa to promote cell migration and proliferation in tissue to close the wound.

[0114] In some embodiments, at least 80 percent of the protein fragments have a molecular weight between about 10 kDa and 85 kDa. In some embodiments, at least 50 percent of the protein fragments have a molecular weight between about 18 kDa and 60 kDa. In some embodiments, at least 85 percent of the protein fragments have a molecular weight greater than about 12 kDa. In some embodiments, at least 90 percent of the protein fragments have a molecular weight greater than about 10 kDa.

[0115] In certain embodiments, the present invention provides an SDP composition comprising a low molecular weight SDP and a pharma- ceutically acceptable carrier. The low molecular weight SDP can have an average molecular weight of less than 60 kDa.

[0116] In a preferred embodiment, the SDP-4 fraction has an average molecular weight of 15-35 kDa as determined by SDS-PAGE / ImageJ analysis as described above, and a pH of 8.1-8.3, an osmolality of about 23 mOsm, and a viscosity of about 1.5-3 cP at 25° C., each as a 50 mg / mL solution in water.

[0117] In a preferred embodiment, the SDP-4 fraction has an average molecular weight of 15-30 kDa as determined by SDS-PAGE / ImageJ analysis as described above, and as a 50 mg / mL solution in water, a pH of 8.1-8.3, an osmolality of about 23 mOsm, and a viscosity of about 1.5-3 cP at 25° C., respectively.

[0118] In a preferred embodiment, the SDP-4 fraction has an average molecular weight of 15-25 kDa as determined by SDS-PAGE / ImageJ analysis as described above, and as a 50 mg / mL solution in water, a pH of 8.1-8.3, an osmolality of about 23 mOsm, and a viscosity of about 1.5-3 cP at 25° C., respectively.

[0119] In another preferred embodiment, the SDP-4 fraction has an average molecular weight of about 18-22 kDa as determined by SDS-PAGE / ImageJ analysis as described above, and a pH of about 8.1-8.3, an osmolality of about 23 mOsm, and a viscosity of about 1.5-3 cP at 25° C., each as a 50 mg / mL solution in water.

[0120] In some embodiments of the SDP-4 fraction, about 39% of the protein fragments of SDP-4 are in the range of 25 kDa to 50 kDa, about 57.7% of the protein fragments are in the range of 20 kDa to 60 kDa, about 72.1% are in the range of 15 kDa to 85 kDa, about 83.6% are in the range of 10 kDa to 85 kDa, and about 85.3% are in the range of 10 kDa to 100 kDa.

[0121] Various SDP compositions can be prepared to include low molecular weight protein fragments, high molecular weight protein fragments, or combinations thereof. The low molecular weight protein fragments reduce inflammation on diseased tissue surfaces and / or wounds and / or promote cell migration and / or proliferation. The low molecular weight protein fragments are also useful for treating inflamed tissue surfaces resulting from active disease states and / or the presence of wounds or scars. In some cases, it may be useful to apply compositions of low molecular weight protein fragments to enhance the wound healing process. These cases may include wounds acquired on the battlefield during war, surgical wounds of those desiring faster healing, e.g., of infection, or for pain relief, etc. The wound healing process is enhanced by increased cell numbers, decreased inflammatory molecules such as MMP-9, and / or increased epithelial cell proliferation.

[0122] High molecular weight protein fragments can increase cell adhesion to or aid in the formation of basement membranes. In some cases, it may be useful to apply a composition of high molecular weight protein fragments to chronic wounds, suppurating wounds, or wounds that are difficult to heal, such as diabetic ulcers and skin burns. High molecular weight protein fragments may be involved in the quality of wound closure, whereas low molecular weight protein fragments are involved in the rate of wound closure. In some cases, applying carefully selected amounts of low molecular weight and high molecular weight protein fragment compositions may be used for optimal wound healing rate and quality. The wound healing process can be improved by increasing structural proteins such as focal adhesion kinase (FAK) and / or tight junctions between cells, such as zonula occludens (ZO-1) structures.

[0123] Low-average molecular weight fractions such as SDP-4 have certain properties that are different from SDP and high-molecular weight fractions. For example, cellular uptake of SDP depends on the molecular weight of the peptide chain. SDP peptide molecules smaller than about 60 kDa are readily absorbed into cultured cells, more specifically human corneal limbal epithelial (hCLE) cells. SDP molecules larger than 60 kDa are almost excluded from absorption from cultured cells. It is also important to note that SDP molecules do not colocalize with lysosomal-associated membrane protein 1 (LAMP-1), a marker of the lysosomal endocytic degradation pathway. As a result, SDP molecules bind to non-specific cell membrane receptors, and molecules smaller than about 60 kDa appear to be absorbed into hCLE cells. More importantly, SDP molecules are not absorbed by the lysosomal degradation pathway, so they are bioavailable and can elicit biological activity.

[0124] Aqueous SDP formulation The SDP compositions and subfractions described herein can be formulated with water and / or a pharmaceutical carrier, in a specific embodiment, the carrier is acetate buffered saline, for example, in ophthalmic formulations.

[0125] In some embodiments, an ophthalmic composition is provided for the treatment of dry eye syndrome in a human or mammal. The composition provided herein can be an aqueous solution containing an effective amount of SDP to treat dry eye syndrome. For example, the effective amount of SDP in the aqueous solution can be about 0.01% to about 80% by weight of SDP. In other embodiments, the aqueous solution can include about 0.1% to about 10% by weight of SDP, or about 0.5% to about 2% by weight of SDP. In certain particular embodiments, the ophthalmic composition comprises about 0.05% w / v SDP, about 0.1% w / v SDP, about 0.2% w / v SDP, about 0.25% w / v SDP, about 0.5% w / v SDP, about 0.75% w / v SDP, about 1% w / v SDP, about 1.5% w / v SDP, about 2% w / v SDP, about 2.5% w / v SDP, about 5% w / v SDP, about 8% w / v SDP, or about 10% w / v SDP.

[0126] In various embodiments, the ophthalmic formulation may include additional components, such as anti-inflammatory agents, buffers, and / or stabilizers, in an aqueous solution. The anti-inflammatory agent may be, for example, hyaluronic acid (HA), hydroxyethyl cellulose, hydroxypropyl methylcellulose, dextran, gelatin, polyol, carboxymethyl cellulose (CMC), polyethylene glycol, propylene glycol (PG), hypromellose, glycerin, polysorbate 80, polyvinyl alcohol, or povidone. The anti-inflammatory agent may be present, for example, at about 0.01% to about 10% by weight, or at about 0.2% to about 2% by weight. In one specific embodiment, the anti-inflammatory agent is HA. In various embodiments, HA may be present at about 0.2% by weight of the formulation. It is also possible to exclude one or more of these components from the formulation.

[0127] The buffering or stabilizing agent of the ophthalmic formulation can be phosphate buffered saline, borate buffered saline, citrate buffered saline, sodium chloride, calcium chloride, magnesium chloride, potassium chloride, sodium bicarbonate, zinc chloride, hydrochloric acid, sodium hydroxide, disodium edetate, or combinations thereof. One or more of these ingredients can also be excluded from the formulation.

[0128] The ophthalmic formulation may further comprise an effective amount of an antimicrobial preservative. The antimicrobial preservative may be, for example, sodium perborate, polyquaternium-1 (e.g., Polyquad® preservative), benzalkonium chloride (BAK), sodium chlorite, brimonidine, brimonidine purite, polexitonium, or a combination thereof. It is also possible to exclude one or more of these ingredients from the formulation.

[0129] The ophthalmic formulation may also include an effective amount of a vasoconstrictor, an antihistamine, or a combination thereof. The vasoconstrictor or antihistamine may be naphazoline hydrochloride, ephedrine hydrochloride, phenylephrine hydrochloride, tetrahydrozoline hydrochloride, pheniramine maleate, or a combination thereof. One or more of these ingredients may also be excluded from the formulation.

[0130] In one embodiment, the ophthalmic formulation can include an effective amount of SDP as described herein in combination with water and one or more ophthalmic ingredients. The ophthalmic ingredients can be, for example, a) polyvinyl alcohol; b) PEG and hyaluronic acid; c) PEG and propylene glycol, d) CMC and glycerin; e) propylene glycol and glycerin; f) glycerin, hypromellose and PEG; or a combination of any one or more of the foregoing ingredients. The ophthalmic formulation can include one or more inactive ingredients, such as HP-guar, borate, calcium chloride, magnesium chloride, potassium chloride, zinc chloride, etc. The ophthalmic formulation can also include one or more ophthalmic preservatives, such as sodium chlorite (Purite® preservative (NaClO2), polyquad, BAK, EDTA, sorbic acid, benzyl alcohol, etc.

[0131] Ophthalmic ingredients, inactive ingredients, and preservatives can be present at about 0.1% to about 5% w / v, for example, about 0.15%, 0.2%, 0.25%, 0.3%, 0.4%, 0.5%, 0.75%, 1%, 1.5%, 2%, 2.5%, or 5%, or in the range between any two of the aforementioned values.

[0132] SDP is highly stable in water, with a shelf-life solution stability that is more than twice as long as native silk fibroin in solution. For example, SDP is highly stable in water, with a shelf-life solution stability that is more than 10 times as long as native silk fibroin in solution. The SDP material does not gel when in aqueous solution, even when sonicated at a 5% (50 mg / mL) concentration. In other embodiments, the SDP material does not gel when in aqueous solution, even when sonicated at a 10% (100 mg / mL) concentration.

[0133] Ophthalmic aqueous formulations The present disclosure also provides certain ophthalmic and / or aqueous formulations that can be used generally, for example, to treat conditions related to the eye. Applicant has found that the use of certain ingredients in ophthalmic formulations, such as an acetate buffer system and low pH levels (neutral pH levels or lower), is surprisingly effective in treating dry eye disease. Additionally, Applicant has found that the formulations disclosed herein are also effective in stabilizing proteins in solution for unexpectedly long periods of time while simultaneously exhibiting low levels of particulates. It has been determined that the use of certain combinations of buffers, osmotic agents, and surfactants is not only surprisingly effective in treating dry eye disease, but can also extend the use of certain proteins at room temperature without degradation of the protein or loss of efficacy of the protein.

[0134] Thus, exemplary formulations include one or more buffering agents, surfactants, and one or more osmotic agents. In some embodiments, the formulations may also include a pH level of about 4.5 to about 6.0. The formulations may optionally include proteins that may be stabilized in solution for extended periods of time. In these embodiments, the formulations may, for example, maintain proteins in solution for periods of more than 4 weeks without gelling, and may maintain a microparticle count of 50 particles / mL or less for microparticles having a diameter of 10 micrometers or greater after a storage period of more than 12 weeks at 4° C. to 40° C.

[0135] In some embodiments, the buffering agent may include histidine, acetate, glutamate, or a combination thereof. In yet other embodiments, the formulation includes one or more buffering agents having a final concentration of about 10 millimolar to about 50 millimolar, or about 20 millimolar to about 40 millimolar. In other embodiments, the concentration of each of the one or more osmotic agents in the formulation is about 30 millimolar to about 40 millimolar, or about 35 millimolar.

[0136] In a preferred embodiment, the buffer can comprise about 0.1% to about 1.0% by weight of sodium acetate and about 0.01% to about 0.1% by weight of acetic acid. In another embodiment, the buffer is comprised of about 0.5% to about 2.0% by weight of sodium acetate and about 0.05% to about 1.0% by weight of acetic acid.

[0137] Preferably, the buffering agent (eg, sodium acetate and glacial acetic acid) maintains the pH of the formulation at about 4.5 to about 6.0, about 5.0 to about 6.0, about 5.2 to about 5.8, about 5.3 to about 5.7, or about 5.4, or about 5.5.

[0138] In some embodiments, the osmotic agent in the formulation may be comprised of a monosaccharide, an inorganic salt, or a combination thereof. In additional embodiments, the osmotic agent may be comprised of mannitol, dextrose, sodium chloride, magnesium chloride, or a combination thereof. In other embodiments, the osmotic agent may be comprised of about 0.1% to about 2% glucose by weight and about 0.1% to about 2% magnesium chloride by weight. In yet other embodiments, the osmotic agent may include about 0.01% to about 2% dextrose by weight and about 0.01% to about 2% magnesium chloride by weight. In further embodiments, the osmotic agent may be comprised of about 0.6% to about 0.9% dextrose by weight and about 0.6% to about 0.9% magnesium chloride by weight.

[0139] Certain embodiments of the formulation may also include one or more surfactants. Surfactants include, but are not limited to, non-ionic detergents, i.e., detergents that include molecules with uncharged head groups. Non-ionic detergents include polyoxyethylene (and related detergents) and glycoside compounds (e.g., alkyl glycosides). Alkyl glucosides include octyl β-glucoside, n-dodecyl-β-D-maltoside, β-decyl-maltoside, digitonin, and the like. Examples of polyoxyethylene-based detergents include polysorbates (e.g., polysorbate 40, polysorbate 60, polysorbate 80 (also called TWEEN-40, TWEEN-60, and TWEEN-80, respectively)), TRITON-X series (e.g., TRITON X-100), TERGITOL series detergents (e.g., NP-40), BRIJ series detergents (e.g., BRIJ-35, BRIJ-58, BRIJ-L23, BRIJ-L4, and BRIJ-O10), and PLURONIC series detergents (e.g., BRIJ-20, BRIJ-25, BRIJ-26, BRIJ-27, BRIJ-28, BRIJ-29, BRIJ-30, BRIJ-31, BRIJ-32, BRIJ-33, BRIJ-34, BRIJ-35, BRIJ-36, BRIJ-37, BRIJ-38, BRIJ-39, BRIJ-40, BRIJ-41, BRIJ-42, BRIJ-43, BRIJ-44, BRIJ-45, BRIJ-46, BRIJ-47, BRIJ-48, BRIJ-49, BRIJ-50, BRIJ-51, BRIJ-52, BRIJ-53, BRIJ-54, BRIJ-55, BRIJ-56, BRIJ-57, BRIJ-58, BRIJ-59, BRIJ-60, BRIJ-61, BRIJ-62, BRIJ-63, BRIJ-64, BRIJ-65, BRIJ-65, BRIJ-66, BRIJ-67, BRIJ-68, BRIJ-69, BRIJ-70, BRI F68. Preferably, the surfactant is polysorbate 40, polysorbate 60, or polysorbate 80. In certain preferred embodiments, the surfactant is polysorbate 80. Preferably, the surfactant is present in the formulation having a final concentration of about 0.02% to about 1% w / w, more preferably about 0.02% to about 0.5% w / w. In certain preferred embodiments, polysorbate 80 is present at a final concentration of about 0.01% to about 2.0% or about 0.02% to about 0.5%. In another embodiment, the only surfactant present in the formulation is polysorbate 80.

[0140] In various embodiments, the osmolality of the formulation is about 170 mOsm / kg to about 300 mOsm / kg. In other embodiments, the osmolality is about 160 mOsm / kg to about 200 mOsm / kg, about 175 mOsm / kg to about 180 mOsm / kg, about 180 mOsm / kg to about 200 mOsm / kg, about 200 mOsm / kg to about 250 mOsm / kg, or about 250 mOsm / kg to about 300 mOsm / kg. In a preferred embodiment, the osmolality is about 160 mOsm / kg to about 280 mOsm / kg, or about 175 mOsm / kg to about 185 mOsm / kg.

[0141] In additional embodiments, the formulation is stored in a container comprising glass or polyethylene. In various embodiments, the container is Type I borosilicate glass. In additional embodiments, the container can be a low density polyethylene container. The formulation has been shown to be stable in low density polyethylene containers for more than six months.

[0142] In other embodiments, the storage period or shelf life of the formulation is about 4 months to about 8 months, about 8 months to about 12 months, about 1 year to about 2 years, or more than 2 years from the date of manufacture. In various embodiments, the microparticle count after storage is about 200 / mL, about 150 / mL, about 100 / mL, about 75 / mL, about 45 / mL, about 35 / mL, about 25 / mL, about 20 / mL, about 15 / mL, about 10 / mL, about 5 / mL, or about 1 / mL. In yet other embodiments, the storage temperature is about 10°C to about 30°C, or about 15°C to about 25°C.

[0143] A preferred embodiment of the ophthalmic or aqueous formulation comprises an acetate buffer containing about 0.04% to about 0.1% by weight polysorbate-80, about 0.2% to about 0.3% by weight sodium acetate, and about 0.01% to about 0.03% by weight acetic acid, and an osmotic agent containing about 0.6% to about 0.9% by weight glucose and about 0.6% to about 0.9% by weight magnesium chloride, resulting in a formulation with a pH of 5.2 to 5.8 and an osmolality of 175 mOsm / kg to 185 mOsm / kg.

[0144] Certain preferred embodiments of the ophthalmic or aqueous formulations include one or more surfactants, one or more osmotic agents, and an acetate buffer system comprising about 0.1% to about 1.0% by weight sodium acetate and about 0.01% to about 0.1% by weight acetic acid, the buffer system maintaining the formulation at a pH of 4.5 to 6.0.

[0145] One preferred embodiment of the ophthalmic or aqueous formulation is comprised of 0.1%-1.0% sodium acetate, 0.01%-0.1% acetic acid, 0.1%-2% magnesium chloride, 0.1%-2% glucose, 0.02%-2% polysorbate 80, an osmolality of about 160-200 mOsm / kg, and a pH of about 4.5-6.

[0146] One preferred embodiment of the ophthalmic or aqueous formulation comprises about 0.25 sodium acetate, about 0.01% acetic acid, about 0.75% magnesium chloride, about 0.75% dextrose, about 0.05% polysorbate-80, an osmolality of about 160-200 mOsm / kg, and a pH of about 4.5-6.

[0147] Another preferred embodiment ophthalmic or aqueous formulation is comprised of 0.1%-1.0% sodium acetate, 0.01%-0.1% acetic acid, 0.1%-2% magnesium chloride, 0.1%-2% glucose, 0.02%-2% polysorbate 80, an osmolality of about 160-200 mOsm / kg and a pH of about 4.5-6.

[0148] Another preferred ophthalmic or aqueous formulation comprises about 0.25% sodium acetate, about 0.01% acetic acid, about 0.75% magnesium chloride, about 0.75% glucose, about 0.05% polysorbate-80, with an osmolality of about 180-190 mOsm / kg and a pH of 5.2-5.7.

[0149] Another preferred embodiment of the ophthalmic or aqueous formulation consists essentially of 0.1%-1.0% sodium acetate, 0.01%-0.1% acetic acid, 0.1%-2% magnesium chloride, 0.1%-2% dextrose, 0.02%-2% polysorbate 80, an osmolality of about 160-200 mOsm / kg, and a pH of about 4.5-6.

[0150] In a more preferred embodiment, the ophthalmic or aqueous formulation consists essentially of about 0.25% sodium acetate, about 0.01% acetic acid, about 0.75% magnesium chloride, about 0.75% glucose, about 0.05% polysorbate-80, an osmolality of about 180-190 mOsm / kg and a pH of 5.2-5.7.

[0151] In certain embodiments, the ophthalmic or aqueous formulation is capable of stabilizing proteins in solution for extended periods of time, for example, the formulation can maintain proteins in solution for periods of more than 4 weeks without gelling, if present, and the formulation can maintain a particulate count of 50 mL or less after a storage period of more than 12 weeks at 4° C. to 40° C. for particulates having a diameter of 10 micrometers or greater.

[0152] In various embodiments, the wt % of protein in the formulation is from about 0.01% to about 15%. In additional embodiments, the wt % of protein is from about 0.1% to about 5%, or from about 1% to about 3%.

[0153] In certain embodiments, the protein included in the ophthalmic or aqueous formulation is a hydrophobic protein. When referring to a hydrophobic protein, the protein may have a "net" hydrophobicity, which is understood to mean that the protein as a whole is more hydrophobic than hydrophilic. Net hydrophobicity is determined using the hydrophobicity index of amino acids. For example, each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge characteristics: isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine / cystine (+2.5), methionine (+1.9), alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). In this example, the more positive the value, the more hydrophobic it is (see, e.g., Kyte et al., A simple method for displaying the hydropathic character of a protein., J. Mol. Biol. (1982) 157(1):105-32, incorporated herein by reference).

[0154] A hydrophobic protein is one that has a positive total hydrophobicity index after the following procedure: converting each amino acid of the polypeptide chain to its respective index value and adding up the values ​​to obtain the total hydrophobicity index. The hydrophobic / non-hydrophobic nature of polypeptides and peptides can be determined as well. It is understood that certain proteins and polypeptides have regions that are hydrophobic and these regions interfere with the analysis or utility of the molecule, for example, MALDI MS. In these cases, the hydrophobicity index for the region is of interest and is determined. In some cases, the region comprises consecutive amino acids, and in other cases, the region comprises a hydrophobic surface resulting from higher order folding (e.g., tertiary structure) of the polypeptide chain.

[0155] In some embodiments, the protein is a small fibrous protein (i.e., has little to no tertiary structure) having an average molecular weight of about 10 kDa to about 50 kDa, about 10 kDa to about 35 kDa, 15 kDa to about 35 kDa, about 15 kDa to about 30 kDa, about 15 kDa to about 25 kDa, about 16 kDa to about 23 kDa, or about 18 kDa to about 22 kDa. In some embodiments, the protein comprises less than 8% serine amino acid residues. In other embodiments, the protein is composed of less than 7.5% serine amino acid residues, less than 7% serine amino acid residues, less than 6.5% serine amino acid residues, or less than 6% serine amino acid residues.

[0156] In some embodiments, the protein comprises greater than 46% glycine amino acids, relative to the total amino acid content of the protein, hi other embodiments, the protein comprises greater than 46.5% glycine amino acids, greater than 47% glycine amino acids, greater than 47.5% glycine amino acids, or greater than 48% glycine amino acids.

[0157] In some embodiments, the protein comprises greater than 30% alanine amino acids, relative to the total amino acid content of the protein, hi other embodiments, the protein is composed of greater than 30.5% alanine amino acids, greater than 31% alanine amino acids, greater than 31.5% alanine amino acids, greater than 32% alanine amino acids, greater than 33% alanine amino acids, or greater than 33.5% alanine amino acids.

[0158] In certain embodiments, the protein is composed of 46.5%-48% glycine amino acids and 30%-33.5% alanine amino acids, based on the total amino acid content of the protein.

[0159] In other embodiments, the protein contains greater than 46% glycine amino acids and greater than 30% alanine amino acids relative to the total amino acid content of the protein.

[0160] Exemplary proteins for use in the formulations can be characterized based on their characteristics in solution. For example, when an exemplary protein is present, the resulting formulation may have an aqueous viscosity of less than 4 cP as a 10% w / w solution in water. In various embodiments, the formulation has an aqueous viscosity of less than 10 cP as a 15% w / w solution in water, or less than 10 cP as a 24% w / w solution in water.

[0161] In a preferred embodiment, the protein is a fibroin-derived protein (e.g., SDP-4). In various embodiments, the wt% of the fibroin-derived protein in the formulation is about 0.01% to about 15%. In additional embodiments, the wt% of the fibroin-derived protein is about 0.1% to about 5%, or about 1% to about 3%. Thus, in a preferred embodiment comprising a protein, the disclosure provides a formulation comprising a fibroin-derived protein, wherein the primary amino acid sequence of the fibroin-derived protein composition differs from that of native fibroin by at least 4% in terms of the absolute value of the sum of the differences in amino acid content of serine, glycine, and alanine; the cysteine ​​disulfide bond between the fibroin heavy protein chain and the fibroin light chain of the fibroin-derived protein is reduced or eliminated; and the cysteines of the fibroin-derived protein composition are reduced or eliminated. The protein composition has a serine content that is reduced by 25% or more compared to native fibroin, where the serine content is at least about 5%; and the average molecular weight of the fibroin-derived protein composition is less than 35 kDa and greater than 15 kDa; and a buffer, polysorbate-80 and one or more osmotic agents; wherein the formulation has a pH of 4.5 to 6.0 and, for microparticles having a diameter of 10 micrometers or greater, has a microparticle count of 50 / mL or less after a storage period of more than 12 weeks at 4°C to 40°C.

[0162] In various other embodiments, the fibroin derived protein composition is Silk Derived Protein-4 (SDP-4) having an average molecular weight of about 15 kDa to about 35 kDa, or about 18 kDa to about 22 kDa, and the pH of the formulation is 5.2 to 5.8, in other embodiments, the pH is about 5.0 to about 6.0.

[0163] Certain preferred embodiments are aqueous formulations for use in treating eye-related conditions that stabilize proteins in solution containing about 0.1% to about 3% by weight, wherein the primary amino acid sequence of the fibroin-derived protein differs from native fibroin by at least 6% in terms of the absolute value of the difference in combined amino acids from native fibroin, wherein the primary amino acid sequence of the fibroin-derived protein differs from native fibroin by at least 6% in terms of the absolute value of the total difference in amino acid content of serine, glycine, and alanine, wherein cysteine ​​disulfide bonds between the fibroin heavy protein chain and the fibroin light protein chain of the fibroin-derived protein are reduced or eliminated, and wherein the fibroin-derived protein comprises greater than 46% glycine amino acids and greater than 30% alanine amino acids. The fibroin-derived protein has a serine content that is reduced by 40% or more compared to native fibroin protein, such that the fibroin-derived protein contains less than 8% serine amino acids; and the average molecular weight of the fibroin-derived protein is about 15 kDa to about 35 kDa; and comprises polysorbate-80, about 10 mM to about 50 mM acetate buffer, and an osmotic agent; wherein the formulation has a pH of 5.2 to 5.8, an osmolality of 175 mOsm / kg to 185 mOsm / kg, and a particulate count of 50 mL or less after a storage period of more than 12 weeks at 4° C. to 40° C. for particulates having a diameter of 10 micrometers or greater.

[0164] In various embodiments, the acetate buffer is comprised of about 0.2% to about 0.3% sodium acetate and about 0.01% to about 0.03% acetic acid by weight. In other embodiments, the osmotic agent comprises about 0.6% to about 0.9% glucose and about 0.6% to about 0.9% magnesium chloride by weight. In additional embodiments, the weight percent of polysorbate-80 is about 0.05% to about 0.1%.

[0165] Additionally, the present disclosure provides a method of treating an ophthalmic disease comprising administering to a subject having the ophthalmic disease an effective amount of a formulation disclosed herein, thereby treating the ophthalmic disease. In some embodiments, the ophthalmic disease is dry eye syndrome.

[0166] In view of the above, the present disclosure provides the following embodiments. 1. An ophthalmic formulation comprising one or more buffers, a surfactant, and one or more osmotic agents, characterized in that the formulation has a pH of 4.5-6.0, the formulation is capable of maintaining proteins in solution for greater than 4 weeks without gelling, and for microparticles 10 μm or greater in diameter, maintains a particle count of 50 mL or less after a storage period of greater than 12 weeks at 4° C.-40° C. The formulation may or may not include a protein composition such as SDP-4. 2. The formulation of clause 1, wherein the osmolality of the formulation is from about 160 mOsm / kg to about 280 mOsm / kg. 3. The formulation of clause 2, wherein the osmolality of the formulation is from about 175 mOsm / kg to about 185 mOsm / kg. 4. The formulation of clause 1, wherein the one or more buffering agents comprise histidine, acetate, glutamate, or a combination thereof. 5. The formulation of claim 4, wherein the formulation has a buffer concentration of about 10 millimolar to about 50 millimolar, or the concentration of each of the one or more osmotic agents in the formulation is about 30 millimolar to about 40 millimolar. 6. The formulation of paragraph 4, wherein the one or more buffering agents include about 0.1% to about 1.0% by weight sodium acetate and about 0.01% to about 0.1% by weight acetic acid. 7. The formulation of paragraph 1, wherein the one or more osmotic agents comprises a monosaccharide, an inorganic salt, or a combination thereof. 8. The formulation of clause 7, wherein the one or more osmotic agents include mannitol, dextrose, sodium chloride, magnesium chloride, or a combination thereof. 9. The formulation of clause 7, wherein the one or more osmotic agents include about 0.10% to about 2.0% by weight of glucose and about 0.10% to about 2.0% by weight of magnesium chloride. 10. The formulation described in item 1, wherein the surfactant is polysorbate 40, polysorbate 60, or polysorbate 80. 11. The formulation of claim 1, wherein the weight percent of the surfactant is about 0.02% to about 1.0%. 12. The formulation of clause 10, wherein the surfactant is polysorbate 80 present in a weight percent of about 0.02% to about 0.5%. 13. The formulation according to clause 1, wherein the formulation has a pH of 5.2 to 5.8 and an osmolality of 175 mOsm / kg to 185 mOsm / kg. 14. The formulation of any one of clauses 1-13, wherein the formulation further comprises a protein, and the weight percent of protein in the formulation is from about 0.01% to about 3%. 15. The formulation described in paragraph 14, wherein the protein is a hydrophobic protein. 16. The formulation described in clause 15, wherein the primary amino acid sequence of the hydrophobic protein comprises about 33% alanine and about 48% glycine. 17. The formulation of paragraph 14, wherein the protein is a fibroin-derived protein comprising: the primary amino acid sequence of the fibroin-derived protein composition differs from that of native fibroin by at least 4% in terms of the absolute value of the total difference in amino acid content of serine, glycine, and alanine, and the cysteine ​​disulfide bonds between the fibroin heavy and fibroin light protein chains of the fibroin-derived protein are reduced or eliminated; the fibroin-derived protein has a serine content that is reduced by 25% or more compared to native fibroin, wherein the serine content is at least about 5%; and the average molecular weight of the fibroin-derived protein in the formulation is less than 35 kDa and greater than 15 kDa. 18. An aqueous formulation comprising about 0.04% to about 0.1% by weight polysorbate-80; an acetate buffer comprising about 0.2% to about 0.3% by weight sodium acetate and about 0.01% to about 0.03% by weight acetic acid; and an osmotic agent comprising about 0.6% to about 0.9% by weight glucose and about 0.6% to about 0.9% by weight magnesium chloride, wherein the formulation has a pH value of 5.2 to 5.8 and an osmotic pressure value of 175 mOsm / kg to 185 mOsm / kg. 19. The aqueous formulation of clause 18, further comprising a protein having a weight percent of about 0.01% to about 3%. 20. The aqueous formulation of clause 19, wherein the protein is a hydrophobic protein having an average molecular weight of less than 35 kDa and more than 15 kDa. 21. An aqueous formulation according to clause 19 or 20, wherein the formulation has a particulate count of 50 / mL or less for particulates having a diameter of 10 micrometers or more after a storage period of more than 12 weeks at 4°C to 40°C. 22. An aqueous formulation consisting essentially of an acetate buffer comprising polysorbate-80 (about 0.04 wt% to about 0.1 wt%), sodium acetate (about 0.2 wt% to about 0.3 wt%), and acetic acid (about 0.01 wt% to about 0.03 wt%), and an osmotic agent comprising about 0.6 wt% to about 0.9 wt% glucose and about 0.6 wt% to about 0.9 wt% magnesium chloride. The formulation has a pH of 5.2 to 5.8 and an osmotic pressure of 175 mOsm / kg to 185 mOsm / kg. 23. An ophthalmic formulation comprising one or more surfactants; one or more osmotic agents; and an acetate buffer system comprising about 0.1% to about 1.0% by weight sodium acetate and about 0.01% to about 0.1% by weight acetic acid, wherein the buffer system maintains the formulation at a pH of 4.5 to 6. The formulation is capable of maintaining protein in solution for a period of greater than 4 weeks without gelling, and the formulation is capable of maintaining a particulate count of 50 mL or less after a storage period of greater than 12 weeks at 4° C. to 40° C. for particulates having a diameter of 10 micrometers or greater when the protein is added to the ophthalmic formulation. 24. The ophthalmic formulation according to item 23, wherein the one or more surfactants is polysorbate 80. 25. The ophthalmic formulation of clause 23, further comprising a protein, the protein being a hydrophobic protein having an average molecular weight of less than 35 kDa and more than 15 kDa. 26. An ophthalmic formulation consisting essentially of one or more surfactants; one or more osmotic agents; and an acetate buffer system comprising about 0.1% to about 1.0% by weight sodium acetate and about 0.01% to about 0.1% by weight acetic acid, wherein the buffer system maintains the formulation at a pH of 4.5 to 6.0. 27. An ophthalmic formulation consisting essentially of a silk fibroin derived protein; one or more surfactants; one or more osmotic agents; and an acetate buffer system comprising about 0.1% to about 1.0% by weight sodium acetate and about 0.01% to about 0.1% by weight acetic acid, wherein the buffer system maintains the formulation at a pH of 4.5 to 6, wherein the formulation is capable of maintaining the protein in solution for greater than four weeks without gelling, and wherein the formulation is capable of maintaining a particulate count of greater than 50 / mL after a storage period of greater than 12 weeks at 4° C. to 40° C. for particulates having a diameter of 10 micrometers or greater when the protein is added to the ophthalmic formulation. 28. An ophthalmic formulation as described in clause 27, wherein the silk-derived protein has an average molecular weight of about 35 kDa to about 15 kDa. 29. The ophthalmic formulation according to clause 27, wherein the one or more surfactants is polysorbate 80. 30. A method for treating an ophthalmic disease, comprising administering to a subject having the ophthalmic disease an effective amount of the combination described in any one of items 1 to 29, thereby treating the ophthalmic disease. 31. The method according to claim 30, wherein the ophthalmic disease is dry eye syndrome.

[0167] treatment The present invention provides for the use of SDP in a formulation to reduce inflammation, for example inflammation on or within the human cornea. Such reduction in inflammation has been demonstrated in both in vitro and in vivo experimental models. Specifically, work has been done to show that SDP suppresses inflammation in a human cornea model by inhibiting NF-κB-related cell signaling pathways, known to be the driving force behind inflammation in the body, one specific example being dry eye disease. Inhibiting these pathways has been shown to ultimately reduce gene expression and tissue retention of MMP-9, known to be the cause of dry eye and ocular inflammation.

[0168] Thus, the present invention provides a method for reducing inflammation and treating wounds, including corneal wounds, comprising administering SDP to a subject site. The method can include administering a formulation comprising a composition of silk-derived protein (SDP), or a molecular fraction thereof, to inflamed tissue, such as wounded live animal tissue. In some embodiments, the subject has an ocular condition that results in inflamed tissue, such as dry eye disease. In some embodiments, the wound is an ocular wound, a surgical wound, an incision, or an abrasion. The ocular wound can be, for example, a corneal wound.

[0169] SDP and SDP-4 can therefore be used to treat and / or reduce inflammation caused by conditions such as wounds, infections, or diseases. Examples of such conditions include ocular wounds, surgical wounds, incisions, or abrasions. In some cases, inflammation is caused by ocular diseases, such as dry eye disease or syndrome, corneal ulcers, corneal erosions, corneal abrasions, corneal degeneration, corneal perforations, corneal scars, epithelial defects, keratoconjunctivitis, idiopathic uveitis, corneal transplants. Age-related macular degeneration (AMD, wet or dry), diabetic eye disease, blepharitis, glaucoma, ocular hypertension, postoperative eye pain and inflammation, posterior segment neovascularization (PSNV), proliferative vitreoretinopathy (PVR), cytomegalovirus retinitis (CMV), endophthalmitis, choroidal neovascularization (CNVM). Ocular symptoms of vascular occlusive diseases, allergic eye diseases, tumors, retinitis pigmentosa, eye infections, scleritis, ptosis, eye pain, mydriasis, neuralgia, scarring ocular surface diseases, eye infections, inflammatory eye diseases, ocular surface diseases, corneal diseases, retinal diseases, systemic diseases, including genetic eye diseases, eye tumors, elevated intraocular pressure, herpes infections, putiligium (scleral tumors), injuries sustained on the ocular surface, eye pain and inflammation after photorefractive keratotomy, corneal or chemical burns, scleral injuries, corneal injuries, and conjunctival injuries. In some embodiments, the inflammation and / or ocular condition is caused by aging, autoimmune conditions, trauma, infection, degenerative diseases, endothelial dystrophy, and / or surgery. In one embodiment, SDP or SDP-4 is used in a formulation for treating dry eye syndrome.

[0170] The following examples are intended to illustrate the above invention and should not be construed as narrowing its scope. Those skilled in the art will readily recognize that the examples suggest many other ways in which the invention can be practiced. It should be understood that numerous variations and modifications can be made while remaining within the scope of the invention.

[0171] example Example 1. Preparation of an over-the-counter anti-inflammatory eye drop formulation Eye drop compositions can be prepared that utilize the therapeutic properties of SDP to treat the ocular system for disease or injury. SDP molecules can be isolated based on molecular weight, or can be used as a whole composition. A composition of protein molecules with a low average molecular weight, such as less than about 35 kDa and more than about 15 kDa, can be prepared and is called SDP-4. A second composition of protein molecules can also be prepared that includes all the molecular weights of the SDP composition or molecules of about 40 kDa or more. Each composition contains water, at least one buffer or buffer system (e.g., phosphate buffered saline (PBS), citrate, borate, Tris, 4-(2-hydroxyethyl)-1-piperazinesulfonic acid (HEPES)), optionally at least one preservative (e.g., perborate, benzalkonium chloride (BAK)), and optionally at least one additional excipient, surfactant, stabilizer or salt (e.g., sulfanilic acid, trehalose, glycerin, ethylenediaminetetraacetic acid (EDTA), polyethylene glycol (PEG), mannitol, polysorbate, sodium chloride (NaCl), magnesium chloride (MgCl2), calcium chloride (CaCl2), or lithium bromide (LiBr)).

[0172] The ophthalmic formulation comprising the first composition can be applied as a treatment product to patients with dry eye disease, wounds, or surgical wounds (e.g., after refractive surgery or cataract surgery) in otherwise healthy patients. The disease or wound can be monitored over time for inflammation and wound closure rates, as well as patient comfort and pain assessments. The second composition can be used as a protein excipient in commercial products, such as artificial tear drops, to help increase the wettability, spread, and patient comfort of the formulation.

[0173] Exemplary eye drop formulations include SDP-4 or SDP in concentrations as low as 0.1 wt. / vol. to as high as 10 wt. / vol. SDP-4 or SDP. The SDP-4 or SDP material would be dissolved in purified water and a buffer system such as citrate buffer, Tris buffer, PBS buffer, borate buffer, etc., would be created in concentrations from 1 mmol to 1000 mmol. The formulation may further include excipient components. Surfactants such as polysorbates can be added in concentrations ranging from 0.01% to 0.1% wt. / vol. Stabilizing sugar molecules such as trehalose, dextrose, or sucrose can be added in concentrations ranging from 10 mmol to 500 mmol. As an ocular lubricant, a quenching molecule such as PEG, carboxymethylcellulose, hypromellose, hydroxypropylmethylcellulose, or glycerin can be added at a concentration ranging from 0.1% to 2.0% wt. / vol. Also, to reduce intermolecular interactions and stabilize the formulation, a salt such as NaCl, MgCl2, CaCl2, or LiBr can be added at a concentration ranging from 10 mmol to 500 mmol. As a stabilizer, an amino acid molecule can be added at a concentration ranging from 10 mmol to 500 mmol, such as L-glutamine or L-arginine. As a stabilizer, a chelating agent such as EDTA can be added at a concentration of 0.01% to 0.1 wt. / vol. Antimicrobial agents can be added at a concentration of up to 0.015 wt. / vol., such as perborate or BAK.

[0174] Table 1 below lists several exemplary base formulations that have been produced containing SDP-4 and / or SDP molecules, to which additional additives or excipients can be added to enhance the formulation applications described above.

[0175] [Table 1]

[0176] SDP, or SDP fractions such as SDP-4, can also be added to known ophthalmic formulations, such as over-the-counter and pre-formulated eye drops and ointments, to improve wettability and patient comfort. Examples of eye drops to which SDP or SDP-4 can be added include brimonidine tartrate, brimonidine tartrate / timolol maleate, alcaftadine, bimatoprost, cyclosporine, gatifloxacin, ketorolac tromethamine, or lifitegrast eye drops. Examples of other formulations to which SDP or SDP-4 may be added are described in U.S. Patent Nos. 5,468,743; 5,880,283; 6,333,045; 6,562,873; 6,627,210; 6,641,834; 6,673,337; 7,030,149; 7,320,976; 7,323,463; 7,351,404; 7,388,029; 7,642,258; 7,842,714; 7,851,504; 8,008,338; 8,038,988; 8,101,161; 8,133,890; 8,207,215; 8,263,054; 8,278,353; 8,299,118; 8,309,605; 8,338,479;8,354,409; 8,377,982; 8,512,717; 8,524,777; 8,541,463; 8,541,466; 8,569,367; 8,569,370; 8,569,730; 8,586,630; 8,629,111; 8,632,760;8,633,162; 8,642,556; 8,648,048; 8,648,107; 8,664,215; 8,685,930; 8,748,425; 8,772,338; 8,858,961; 8,906,962; 9,248,191, and U.S. Patent Nos. 7,314,938; 7,745,460; 7,790,743; 7,928,122; 8,084,047; 8,168,655; 8,367,701; 8,592,450; 8,927,574; 9,045,457; 9,085,553; 9,216,174; 9,353,088; and 9,447,077.

[0177] Example 2. Preformulation studies Bombyx mori silkworm cocoons were purchased from Shanghai Yu Yuan Company. Raw silk was extracted using a 0.3% wt. / wt. aqueous solution. It was extracted with Na2CO3 (JT Baker, USP Grade) solution at 95°C for 75 min and rinsed thoroughly with purified water (SilkTech Biopharmaceuticals) for 20 min. The rinse cycle was then repeated three more times to ensure that all residual Na2CO3 and extracted glue-like sericin proteins were washed away. The degummed extracted silk fibers were then pressed to remove excess water and dried in a convection oven at 70°C for 16 h.

[0178] The dried extracted silk fibers were then solubilized in 54% wt / wt Lithium Bromide (LiBr) solution (FMC Lithium, Inc) at a ratio of 4xLiBr volume per gram of extracted fibers in a process called Reaction. This process was carried out at a temperature of 121°C and 15 psi for various solubilization times to obtain an intermediate solution called SDP / LiBr intermediate. This intermediate solution was then fractionated using a tangential flow filtration (TFF) 30kDa Sartorius Hydrosart cutoff filter, retaining all fractions below 30kDa. This fraction was then filtered using a TFF 10kDa Sartorius Hydrosart cutoff filter, retaining all fractions above 10kDa. The result was Silk Derived Protein-4 (SDP-4). All Sartorius Hydrosart cutoff filters were purchased from Sartorius Stedim.

[0179] During the initial formulation development phase (Tables 2-6), stock solutions of various buffers, salts, sugars, and surfactants were made. These stock solutions were then added directly to SDP-4 (reacted for 30 minutes) and diluted with purified water until the desired concentrations of excipients and SDP-4 were reached. The solutions were mixed until completely dissolved, filtered through a 0.2 μm polyethersulfone filter (VWR), and placed into a 50 mL polypropylene conical (VWR). The formulation containers were then placed in a stability chamber at 40°C and 75% relative humidity to monitor stability. Table 2 below lists the chemicals and manufacturers for the initial formulation development. Each item in the preliminary formulation study (Tables 3-6) was evaluated by qualitative analysis. The acceptance criteria were no gelling and essentially no visible particulates. For each item in the preliminary formulation study, it was rejected during the preliminary formulation study screening process due to not meeting the acceptance criteria.

[0180] [Table 2]

[0181] [Table 3]

[0182] [Table 4]

[0183] [Table 5]

[0184] [Table 6]

[0185] [Table 7]

[0186] Example 3. Effect of pH and temperature on SDP-4 The dried extracted fibers were reacted in a benchtop reactor for 30 or 200 minutes. The intermediates were further processed using a TFF with 30 kDa and 10 kDa Sartorius Hydrosart filters to obtain SDP-4 (30 min reaction) and SDP-4 (200 min reaction). These two specimens were then titrated to the desired pH using 1 M hydrochloric acid (Lab Chem). The samples were then diluted to a concentration of 1% wt. / wt. They were then filtered using polyethersulfone filters (VWR) and dispensed into 50 mL polypropylene conicals. These conicals containing SDP-4 at various reaction times and pH were placed in a stability chamber under conditions of 4 °C, 25 °C, and 40 °C. At the specified time points, samples were removed from the stability chamber and analyzed for pH and particulate matter using an Orion Versa Star pH meter and a Coulter Particulate Counter, respectively. Figure 1 shows a summary graph of SDP-4 reacted for 30 min under various pH and temperature conditions, and Figure 2 shows a summary graph of SDP-4 reacted for 200 min under these same conditions. Comparing the 30 and 200 min reactions, it can be seen that the formation of aggregates is significantly reduced with increasing SDP reaction time.

[0187] Aggregation of SDP-4 protein occurs below pH 5.0. In this study, a combination of centrifugation and filtration was used to remove the flocculants. The retained SDP-4 supernatant and filtrate were then used for the study. As shown in Figure 2, within the pH range of 4.5-6.0, the number of particles formed was the lowest. Conditions above pH 5.0 and 40 °C resulted in an increase in both subvisible and visible particles. In summary, this study shows that the physical stability of SDP-4 decreases with increasing pH, increasing storage temperature, and decreasing reaction time.

[0188] Example 4. Effect of pH and temperature on citrate buffer formulations Silk Derived Protein-4 (30 min or 200 min benchtop reaction) was added to citrate buffer. Citrate buffer is composed of citric acid (VWR) and sodium citrate (VWR). The desired pH was obtained by varying the ratio of citric acid to sodium citrate. SDP-4 was added to citrate buffer and then diluted with purified water to reach a final concentration of 50 mM citrate buffer and 1.0% wt. / wt. SDP-4 concentration. The formulation was then filtered using a polyethersulfone filter (VWR) and dispensed into 50 mL polypropylene conicals (VWR). These conicals containing SDP-4 with various reaction times and citrate formulation pH were placed in a stability chamber under 40°C conditions. After 2 weeks, the samples were measured for particulates using a Coulter particulate counter. Figure 3 shows the effect of reaction time and pH on particle formation, and Figure 4 shows the effect of storage temperature on particle formation. These studies show that in citrate buffered formulations, the physical stability of SDP-4 decreases with increasing pH, increasing storage temperature, and decreasing reaction time.

[0189] Example 5. Compatibility of SDP-4 and container closure systems Development batches of SDP-4 were manufactured at SilkTech Biopharmaceuticals and stored in various container closures to investigate the impact of compatibility of SDP-4 with container closures.

[0190] Three types of container closures were selected for this study: low-density polyethylene (LDPE), glass, and polypropylene (PP). The container closures were washed with purified water to remove any particles from the manufacturing process. A development batch of SDP-4 with a concentration of 5.79% wt. / wt was prepared by adding 2954.6 g of purified water to 617.0 g of SDP-4 and diluting with purified water. To analyze the headspace, the containers were filled with serological pipettes at 50% and 100% of the volume. After filling, the samples were stored under accelerated conditions of 40 °C and 75% relative humidity (RH). After 2 weeks, the appearance and particulate matter (using the Coulter method) of the samples were measured. Table 8 details the materials and equipment used.

[0191] [Table 8]

[0192] Table 9 shows the results after 2 weeks at 40° C. and 75% RH. A summary of the results is shown in Table 10. Figure 5 shows the particle count results for the average subvisible particle count (>10 μm) per mL at 50% of the container volume.

[0193] [Table 9]

[0194] [Table 10]

[0195] Glass had the lowest number of subvisible particles, and no visible particles or aggregates were observed. Low density polyethylene storage had a higher subvisible particle count compared to solutions stored in glass. No visible particles were visually observed in LDPE due to the opaqueness of the container closure. Polypropylene storage showed the most subvisible visible particles of any container closure. Headspace does not appear to be a factor in particle formation in glass and LDPE.

[0196] Based on the data and observations from the 1% wt. / wt. occurrence SDP-4, glass produced less particle formation than LDPE and polypropylene container closures. Glass was selected as the primary container for SDP-4 with the understanding that stability studies appropriate for the SDP-4 phase would be conducted.

[0197] Example 6. Pre-formulation evaluation and stability screening The stability of preformulations of SDP-4 was evaluated. The osmolality of the solutions was adjusted to 290 mOsm / kg (+10 mOsm / kg) using either sodium chloride or mannitol. A description of the 10x diluent and active formulation (SDP-4 is the active pharmaceutical ingredient, denoted as API-1) is shown in Table 11.

[0198] Diluents were diluted 1:10 with milli-Q water, filtered through a 0.2 μm, 25 mm Acrodisc (Pall p / n 4907), and 20 mL was dispensed into 20 cc clear glass serum vials to serve as controls. 20 x 1 mL of each active formulation was filtered through a 0.2 μm, 25 mm Acrodisc (Pall p / n 4907), dispensed into 20 cc clear glass serum vials, and labeled. 1x diluents and active formulations were placed at 32.5°C to 40°C.

[0199] All samples were checked for appearance at 1, 2, and 4 weeks. For samples showing "Opalescence" or "Turbidity", an additional "%Transmissionance" was performed at 500 nm. For all non-gelled samples, %T500nm was performed at 2 and 4 weeks.

[0200] The acceptance criteria for a successful formulation are that it should be essentially free of gelling, clarification, and visible particles.

[0201] [Table 11]

[0202] [Table 11]

[0203] [Table 11] [Table 11]

[0204] [Table 11] [Table 11] [Table 11]

[0205] One week observations: Solutions containing povidone and either sodium phosphate or tromethamine gelled. Silk Derived Protein-4 (API-1, Figure 11), prepared in sodium phosphate, pH 7.2 buffer and tromethamine, pH 8.1 buffer, were the only samples to exhibit a milky color.

[0206] Two week observations: Solutions containing povidone and sodium phosphate, pH 7.2 or tromethamine pH 8.1 were firmer gels than at 1 week. Histidine, pH 6.1 buffer with povidone gelled. Overall, most solutions started to yellow after 2 weeks. An improvement to the %T 500nm method for assessing turbidity / mildness was established by allowing samples to settle for approximately 4 hours after appearance testing, mixing gently to prevent air bubble formation, and reading the %T @500nm using a quartz cuvette. These procedures allowed for a more robust method for reading the %T @500nm. Some samples thickened from the first week onwards and were monitored to see if they would gel further.

[0207] Four week observations:Solutions containing povidone and histidine pH 6.1, sodium phosphate, pH 7.2, or tromethamine pH 8.1 gelled. Some solutions thickened but did not gel. Solutions formulated with low pH or citrate and polysorbate-80 performed better than povidone. Neither mannitol nor sodium chloride performed better than the others.

[0208] Example 7. Preformulation evaluation using diglycine buffer and excipients Preformulation studies were conducted using a diglycine buffer system containing commonly used ophthalmic excipients and commonly used surfactants to stabilize proteins. Table 12 shows the effect of SDP-4 with polyethylene glycol-40 (PEG-40), diglycine buffer, and various sugars (mannitol, trehalose, sorbitol). Formulations were filtered using a 0.2 μm PES filter to remove particles, stored in Type I borosilicate glass serum vials at 40°C temperature conditions, and monitored for 3 weeks. All formulations failed the screening process due to particle formation.

[0209] [Table 12]

[0210] Additional formulation studies were performed using Tetronic 1107 and a diglycine buffer system. Table 13 shows the effect of Tetronic 1107 with a diglycine buffer system and SDP-4 with glycerol and mannitol. Formulations were filtered using a 0.2 μm PES filter to remove particles, stored in Type I borosilicate glass serum vials at 40°C, and monitored for three weeks. All formulations failed the screening process due to particle formation.

[0211] [Table 13]

[0212] Example 8. Formulation Development Buffer Selection Given the results of Examples 1-4, a selection of three buffers at pH 5.5 were evaluated to achieve the pH requirements of SDP-4. These buffers include histidine, acetate, and glutamate buffers at concentrations of 10 and 50 mM. The acetate buffers were mixed with sodium acetate (VWR) and acetic acid (VWR) in specific ratios to reach the desired pH. The histidine (VWR) and glutamine (VWR) buffers were adjusted using 1 M hydrochloric acid (Lab Chem). Each buffer was adjusted to reach the desired pH value of 5.5. Silk Derived Protein-4 was added to the buffers and diluted with purified water to achieve the desired buffer concentrations of 10 mM and 50 mM. The final concentration of SDP-4 in the formulation was diluted to 1.0% wt. / wt. The formulated SDP-4 was then filtered using a polyethersulfone filter (VWR) and dispensed into Type I glass borosilicate vials (Prince Sterilization). The vials were placed in a stability chamber at 40°C for 8 weeks. Initial and final measurements of pH and particle count were performed using an Orion VersaStar pH meter and a Coulter Particulate Counter. Figure 6 represents the particle count after 8 weeks under storage conditions of 40°C and 75% relative humidity. The glutamate and acetate buffers inhibited particle formation compared to the histidine buffer. All glutamate and acetate buffers were essentially free of visible particles. Figure 7 shows the pH of the formulations initially and after 8 weeks, illustrating the pH drift that occurred in these formulations. While glutamate was insufficient to maintain the pH of SDP-4, the 50 mM acetate and histidine buffers were effective in maintaining the pH of the solution over time. Given the effective buffering capacity and the low particle formation observed, the acetate buffer formulation was selected for subsequent studies. A final buffer concentration of 25 mM was selected as the midpoint of the buffer strengths evaluated, as this met the requirement for maintaining the pH of the formulations.

[0213] Example 9. Effect of osmolarity on SDP-4 formulations A study was conducted to monitor the effect of osmolality on the stability of acetate buffered formulations. Two formulations, containing 25 mM acetate buffer and 1.0% wt. / wt. SDP-4, respectively, were formulated with various levels of mannitol to reach osmolalities of 180 and 290 mOsm / kg. The formulated SDP-4 acetate buffered formulations were filtered using a PES filter to remove any initial particles and dispensed into Type I glass borosilicate vials. The vials were placed in a stability chamber at 40°C and monitored daily. Figure 8 shows the results for these two formulations. From the figure, it can be seen that the formulation with an osmolality of 290 mOsm / kg fails within one day while the formulation with an osmolality of 180 mOsm / kg remains stable for 14 days. The criteria for a formulation to fail is greater than 50 particle counts per mL with particle sizes between 10 and 25 μm. This study showed that the physical stability of SDP-4 formulations depends on the osmolality of the formulation.

[0214] Example 10. Selection of an excitatory substance that increases osmotic pressure The following excipients were considered to increase the osmolality of the formulation: sodium chloride (NaCl), magnesium chloride (MgCl2), mannitol, and dextrose. Results of the initial screen described in Example 5 excluded commonly used excipients including glycerol, povidone, calcium chloride (CaCl2), trehalose, ethylenediaminetetraacetic acid (EDTA), and polyethylene glycol 400 (PEG400).

[0215] The dried extracted fiber was reacted for 240 minutes at the required temperature and pressure in a production-scale reactor. The SDP / LiBr intermediate was fractionated in a benchtop TFF unit to become SDP-4. An acetate buffer containing sodium acetate (VWR) and acetic acid (VWR) was mixed in the specified ratio to achieve the desired acetate buffer pH of 5.4. The precipitate was then added to the acetate buffer, followed by SDP-4. The final concentration of the acetate buffer was 25 mM and the final concentration of SDP-4 was 1.0% wt. / wt. All excipients were added in various amounts to reach a target osmolality of 180 mOsm / kg. The formulation was then filtered using a polyethersulfone filter (VWR) and dispensed into Type I glass borosilicate vials (Prince Sterilization). The vials were placed in stability chambers at 25°C and 40°C and evaluated for particulates using visual testing and a Coulter particulate counter. Table 14 lists the raw materials used and their manufacturers.

[0216] [Table 14]

[0217] Table 15 summarizes the two-week observations of the Type I borosilicate glass formulations. During the visible particle screening of MgCl2, it was identified that no particles formed at 25°C, but at 40°C, it started forming debris and sphere-like particles. The dextrose formulation formed fewer particles than the mannitol formulation at 25°C. Furthermore, it was observed that salt formed fewer aggregates but was susceptible to gelation. Conversely, sugar retarded gelation but formed more aggregates. Hence, the salt and sugar blend is optimal to forestall particle formation and gelation.

[0218] [Table 15]

[0219] Figure 9 shows the formulation of the subvisible particle measurements shown in Table 15. Magnesium chloride and dextrose result in less particle formation compared to sodium chloride and mannitol. The combination of 50% MgCl2 and 50% dextrose results in the least subvisible particle formation.

[0220] Example 11. Selection of surfactants Two official surfactants available for use in ophthalmic solutions, polysorbate-20 and polysorbate-80, were obtained from Croda and evaluated at the concentrations shown in Table 16. All formulations were manufactured using 21.5 mM sodium acetate, 3.5 mM acetic acid, and 1.0% wt. / weight SDP-4 and were stored in Type I borosilicate glass at 40°C / 75% relative humidity for 12 weeks. Analyses were performed for appearance, pH, total protein by UV / Vis, and particulate matter tested by the Coulter method. Table 16 shows the formulations by excipient concentration, and Table 17 shows the results of the screening after 12 weeks. Table 17 shows only those formulations that passed the appearance test (essentially free of visible particles). All of these formulations contain polysorbate-80. The polysorbate-20 formulations formed even more visual particles than the formulations without the surfactant. The results of the screening showed that the formulations containing polysorbate-80 were more consistent with the USP <789> The results showed that SDP-4 formulations pass particulate matter using the standard of 100% and maintain pH and total protein content. Figure 10 shows the results for the formulations without polysorbate-80, and Figure 11 shows the particle count results between the formulations with polysorbate-80 and polysorbate-20. The results of this study showed that surfactants are necessary to maintain the long-term physical stability of SDP-4 formulations. However, the correct surfactant must also be selected. Although polysorbate-20 and polysorbate-80 have very similar chemical structures, polysorbate-80 enhances the stability of SDP-4 formulations by inhibiting particle formation. Polysorbate-20 promotes particle formation.

[0221] [Table 16]

[0222] [Table 17]

[0223] Example 12. Formulation screening of standard ophthalmic buffers Additional formulation studies were conducted to investigate whether other commonly used ophthalmic buffers would yield the same results in inhibiting particle formation in combination with known particle-inhibiting excipients (magnesium chloride, dextrose, polysorbate-80). A selection of three buffers were investigated, including sodium phosphate, citrate phosphate, and Tris hydrochloride.

[0224] Sodium phosphate monobasic monohydrate (JT Baker) and sodium phosphate dibasic 12-hydrate (JT Baker) were mixed in the desired ratio to achieve a pH of 7.0. Citric acid monohydrate and sodium phosphate dibasic were mixed in the desired ratio to achieve a pH of 7.0. Tris hydrochloride (JT Baker) was titrated using sodium hydroxide (VWR) to achieve the desired pH of 7.0. Magnesium chloride hexahydrate and anhydrous dextrose were purchased from JT Baker and mixed into stock solutions. Super Refined Polysorbate 80 was purchased from Croda.

[0225] The order of addition for the formulation was as follows: Polysorbate-80 was added first, then 80% of the water volume, then the buffer stock solution, then magnesium chloride and dextrose, then Silk Derived Protein-4, and finally water.

[0226] The formulation was then filtered using a polyethersulfone filter (VWR) and dispensed into Type I glass borosilicate vials (Prince Sterilization). The vials were placed in a stability chamber at 40° C. and 75% relative humidity and evaluated for particulates using a visual test. The results of the screening are shown in Table 18.

[0227] [Table 18]

[0228] The results of the study show that formulations of sodium phosphate, citrate phosphate, and Tris hydrochloride in combination with known particle-inhibiting excipients (magnesium chloride, dextrose, polysorbate-80) do not inhibit particle formation. Two of the buffers, sodium phosphate and citrate phosphate, quickly formed insoluble particles during titration to the desired pH. Tris hydrochloride failed the appearance screening test after six weeks under storage conditions at 40°C.

[0229] Example 13. Formulation development using standard surfactants Additional formulation studies were conducted to investigate whether other commonly used ophthalmic surfactants would provide the same results in inhibiting particle formation when combined with known particle inhibitors (magnesium chloride, dextrose, acetate). A selection of four surfactants were investigated, including poloxamer 188, poloxamer 407, polyethylene glycol 300, polyethylene glycol 400, and polyethylene glycol 600.

[0230] The order of addition for the formulation was to add 80% of the desired water content, followed directly by the surfactant, magnesium chloride, dextrose, sodium acetate trihydrate, and glacial acetic acid. The formulation was then mixed until all excipients were completely dissolved. Next, Silk Derived Protein-4 was added, and finally the water was added.

[0231] The formulation was then filtered using a polyethersulfone filter (VWR) and dispensed into Type I glass borosilicate vials (Prince Sterilization). The vials were placed in a stability chamber at 40° C. and 75% relative humidity and evaluated for particulates using a visual test. The results of the screening are shown in Table 19.

[0232] [Table 19]

[0233] The results of the study indicate that formulations of poloxamer 188, poloxamer 407, PEG-300, PEG-400, and PEG-600 with other particle-inhibiting excipients (magnesium chloride, dextrose, polysorbate-80) do not inhibit particle formation. In all items in Table 19, the surfactants failed the 1-3 week screening process.

[0234] Example 14. SDP-4 eye drops medicine Dosage Form: Silk-derived protein-4 (SDP-4) sterile topical eye drop drug product (DP) contains SDP-4 drug substance (DS) in a single-use vial. The osmolality agent is adjusted to establish an osmolality of 180 mOsm / kg (± 1%) (Table 21-23).

[0235] [Table 21]

[0236] [Table 22]

[0237] [Table 23]

[0238] Container type and dosage form closure:The DP was supplied in single unit dose (SUD) low density polyethylene (LDPE) vials with fill ranges of 0.512-0.589 g. The DP and vials were manufactured blow-fill-seal (BFS) utilizing a sterile filling process of the DP into BFS vials, allowing for droplet volume sizes of 20 μL-50 μL.

[0239] Pharmaceutical containers and closures types: Sealed SUDs with a total liquid volume of 1 mL were fabricated from LDPE using the blow-fill-seal (BFS) process.

[0240] [Table 24]

[0241] Stability studies were performed on the formulations contained in Tables 21-23. The environmental conditions for the stability studies were 40°C / 75% relative humidity. Initial measurements were made at the time of manufacture and at 6 months. Each formulation was tested for appearance, pH, osmolality, and particulate matter. Tables 25-27 show the results of the stability studies. The formulations have been shown to inhibit particulate formation and maintain solution pH and osmolality under conditions of 40°C / 75% relative humidity in low density polyethylene container closures. The development and summary of the formulation work has resulted in formulations that meet all specifications within the container closures favorable for commercial ophthalmic use under storage conditions that are normally unfavorable for therapeutic proteins.

[0242] [Table 25]

[0243] [Table 26]

[0244] [Table 27]

[0245] Example 15. Treatment of dry eye: Ophthalmic formulations with and without SDP-4The primary objective of this study was to evaluate the safety and efficacy of SDP-4 ophthalmic solution in subjects with DED over a 12-week (84-day) treatment period.

[0246] Study design: This was a phase 2, multicenter, double-masked, randomized, vehicle-controlled, dose-response, parallel-group study designed to evaluate the ocular and systemic safety and efficacy of SDP-4 ophthalmic solution in subjects with moderate to severe DED in both eyes (OU) over a 12-week (84-day) treatment period.

[0247] Subjects were randomized to one of three concentrations (0.1%, 1.0%, and 3.0%) of SDP-4 ophthalmic solution or vehicle in a 1:1:1:1 ratio in parallel groups. All investigational products (IPs) (SDP-4 concentrations and vehicle) were provided in single-use dose (SUD) containers sealed in foil pouches. Subjects, investigators, and all site personnel responsible for conducting study assessments remained masked to treatment allocation.

[0248] IP was administered by topical ocular instillation, 1 drop per eye, twice daily (BID), for 12 weeks (84 days). Both eyes were treated. A 2-week screening / run-in period with vehicle BID preceded the 12-week randomized treatment period.

[0249] Subjects were required to have a Symptom Assessment of Dry Eye (SANDE) total score ≥40 at Visit 1 / Screening and Visit 2 / Day 1 to participate in the study. For subjects with eligible SANDE scores who met all other inclusion / exclusion criteria, the eye with the shorter tear break-up time (TBUT) at Visit 2 / Day 1 was designated as the study eye. If both eyes had the same TBUT score, the eye with the lower Schirmer's test score was designated as the study eye. If both eyes had the same TBUT and Schirmer's test scores, the right eye was designated as the study eye.

[0250] The study consisted of seven clinic visits, two visits during the screening period, and five visits during the treatment visit: Visit 1 (day -14 ± 2 / screening visit), followed by a 2-week run-in period in the BID vehicle, Visit 2 (day 1 / confirmation and randomization visit), Visit 3 (day 7 ± 2), Visit 4 (day 14 ± 2), Visit 5 (day 28 ± 2), Visit 6 (day 56 ± 4) and Visit 7 (day 84 ± 4 / end-of-study assessment).

[0251] If subjects reported persistent dry eye symptoms, the site was permitted to provide subjects with unstored artificial tears (provided by the sponsor), to be used only if necessary. Subjects were to return all used and unused artificial tears at every visit, allowing sites accountability for assessing artificial tear use. Artificial tears were not to be used within 2 hours prior to the study visit.

[0252] Measuring effectiveness Efficacy was measured by assessment of DED symptoms (SANDE total score, individual symptoms rated on a visual analog scale (VAS): itching, foreign body sensation, burning / stinging, vision fluctuation, eye dryness, eye discomfort, photophobia, and eye pain) and signs (TBUT, Schirmer's test [anesthesia], corneal fluorescein staining, conjunctival lissamine green staining, and conjunctival hypertension) (Figure 12). All efficacy assessments were performed at the time points indicated in the schedule of visits and examinations.

[0253] Primary efficacy variable The primary efficacy endpoint (SANDE) was summarized using continuous summary statistics by treatment group and visit. The primary analysis utilized a repeated measures mixed model with the dependent variable being the change from baseline score, treatment group as the fixed effect, baseline score as the covariate, and visit as the repeated measure of subjects. Repeated measures mixed models were used to account for the effect of missing data under the assumption that data were missing at random. Least squares means were used to test each concentration of SDP-4 against vehicle. Sensitivity analyses for the primary endpoint were performed using last observation carried forward (L°F).

[0254] Efficacy analysis At baseline, the mean total SANDE score ranged from 67 to 71 units (on a 0-100 scale). This measure improved (decreased in value) beginning at day 7 in all treatment groups and continued to improve throughout the study. At day 84, the primary outcome measure, the mean reduction in this measure was 25, 30, 25, and 26 in the SDP-4 and vehicle groups of 0.1%, 1.0%, and 3.0%, respectively. See Table 28, Figures 13-15. The mean total SANDE score difference (active vs. vehicle groups) for the patient population that included patients with a starting SANDE score of 70 or greater was not statistically significant until after 28 days (p=0.2839-0.7952, Figure 15A). Thus, the vehicle formulation also provides an effective treatment for dry eye symptoms. However, the Amlisimod 1% formulation was statistically more effective than vehicle for the subpopulation of patients that did not include those with an initial SANDE score of 70 or greater and who showed significant improvement in the overall population (FIG. 15B), indicating that the SDP-4 (Amlisimod) formulation (Table 22) is highly effective in treating dry eye.

[0255] [Table 28] [Table 28]

[0256] Scale: 0-100 (None to Severe) Test statistics and estimates are from restricted maximum likelihood repeated measures mixed models with interaction with treatment group as repeated measures and visit and change from baseline value as covariates using an unstructured covariance structure. (1) Least squares (LS) means and standard errors (SE) by treatment group. (2) Treatment effect: least squares mean (LSM) difference between SDP-4 and vehicle, standard error (SE), and 95% confidence interval (CI). (3) p-value comparing SDP-4 with the vehicle.

[0257] Although particular embodiments have been described above with reference to disclosed embodiments and examples, such embodiments are illustrative only and do not limit the scope of the present invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects, as defined in the following claims.

[0258] All publications, patents, and patent documents are incorporated herein by reference as if individually incorporated by reference. No limitations that are inconsistent with the present disclosure should be understood therefrom. The present invention has been described with reference to various specific preferred embodiments and techniques. However, it should be understood that many variations and modifications can be made while remaining within the spirit and scope of the present invention. In relation to the present invention, the following is further disclosed: [1] (a) a fibroin-derived protein composition, the primary amino acid sequence of which differs from that of native fibroin by at least 4% in terms of the absolute value of the sum of the differences in amino acid content of serine, glycine, and alanine; the cysteine ​​disulfide bonds between the fibroin heavy protein chain and the fibroin light protein chain of the fibroin-derived protein are reduced or eliminated; the serine content of the protein composition is reduced by 25% or more compared to native fibroin; Moreover, the serine content is at least about 5%; and the average molecular weight of the fibroin-derived protein composition is between 15 kDa and 36 kDa; (b) Polysorbate-80; (c) one or more buffering agents; (d) one or more osmotic agents; A formulation comprising: wherein said formulation has a pH of 4.5 to 6.0 and a particle count of 50 / mL or less for particles having a diameter of 10 micrometers or greater after a storage period of more than 12 weeks at 4° C. to 40° C. [2] The formulation of [1], wherein the protein composition comprises greater than 46.5% glycine amino acids, the protein composition comprises greater than 30.5% alanine amino acids, or a combination thereof. [3] The formulation of [1], wherein the protein composition is reduced by more than 40% compared to native fibroin protein, such that the protein composition contains less than 8% serine amino acids. [4] The formulation described in [1], wherein more than 50% of the protein chains of the protein composition have a molecular weight within the range of 10 kDa to 40 kDa. [5] The formulation described in [1], wherein the primary amino acid sequence of the fibroin-derived protein composition differs from that of natural fibroin by at least 6% in terms of the difference in the sum of serine, glycine, and alanine content; the average molecular weight of the fibroin-derived protein is from about 15 kDa to about 30 kDa; and the pH of the formulation is between about 5.2 and about 5.8. [6] The formulation of [1], wherein the fibroin-derived protein composition has an average molecular weight of about 15 kDa to about 25 kDa and the pH of the formulation is 5.3 to 5.7. [7] The formulation described in [1], wherein the fibroin-derived protein composition has an average molecular weight of about 18 kDa to about 25 kDa. [8] The formulation described in [1], wherein the weight percentage of the fibroin-derived protein is about 0.05% to about 10%. [9] The formulation according to [1], wherein the osmolality of the formulation is from about 170 mOsm / kg to about 300 mOsm / kg.

[10] The formulation of [1], wherein the buffer comprises histidine, acetate, citrate, glutamate, or a combination thereof.

[11] The formulation of

[10] , wherein the buffer concentration formed by the buffer is from about 10 millimolar to about 50 millimolar, or the concentration of each of the one or more osmotic agents in the formulation is from about 30 millimolar to about 40 millimolar.

[12] The formulation of

[11] , wherein the buffer comprises about 0.1% to about 1% by weight of sodium acetate and about 0.01% to about 0.1% by weight of acetic acid.

[13] The formulation of [1], wherein the osmotic agent comprises a monosaccharide, an inorganic salt, or a combination thereof.

[14] The formulation of

[13] , wherein the osmotic agent comprises mannitol, dextrose, sodium chloride, magnesium chloride, or a combination thereof.

[15] The formulation of

[14] , wherein the osmotic agent comprises about 0.10% to about 2% by weight of dextrose and about 0.10% to about 2% by weight of magnesium chloride.

[16] The formulation described in [1], wherein the weight percentage of polysorbate-80 is about 0.02% to about 2%.

[17] The formulation of [1], wherein the formulation is stored in a container comprising glass or polyethylene, optionally wherein the container is Type I borosilicate glass or LDPE.

[18] (a) about 0.1% to about 3% by weight of fibroin-derived protein, the primary amino acid sequence of which differs from that of native fibroin by at least 6% in terms of the absolute value of the sum of the differences in amino acid content of serine, glycine, and alanine; a fibroin-derived protein in which the cysteine ​​disulfide bonds between the fibroin heavy protein chain and the fibroin light protein chain are reduced or eliminated; the fibroin-derived protein contains greater than 46% glycine amino acids and greater than 30% alanine amino acids; the serine content of the fibroin-derived protein is reduced by more than 40% compared to native fibroin protein, such that the fibroin-derived protein contains less than 8% serine amino acids; and the average molecular weight of the fibroin-derived protein is from about 15 kDa to about 35 kDa; (b) Polysorbate-80; (c) about 10 millimolar to about 50 millimolar acetate buffer; and (d) one or more osmotic agents: 1. An aqueous formulation comprising: The formulation has a particulate count of 50 / mL or less for particulates 10 micrometers or greater in diameter after a storage period of 12 weeks or greater at 4°C to 40°C; a pH of 5.2 to 5.8; and an osmolality of 175 mOsm / kg to 185 mOsm / kg.

[19] The formulation of

[18] , wherein the acetate buffer comprises about 0.2% to about 0.3% by weight of sodium acetate and about 0.01% to about 0.03% by weight of acetic acid.

[20] The formulation of

[19] , wherein the osmotic agent comprises about 0.6% to about 0.9% by weight of dextrose and about 0.6% to about 0.9% by weight of magnesium chloride hexahydrate.

[21] The formulation according to

[20] , wherein the weight percentage of polysorbate-80 is about 0.01% to about 0.1%.

[22] A method for treating an eye disease, comprising administering to a subject having an eye disease an effective amount of the formulation described in any one of [1] to

[21] , thereby treating the eye disease.

[23] The method according to

[22] , wherein the eye disease is dry eye syndrome.

Claims

1. (a) a fibroin-derived protein composition, the primary amino acid sequence of which differs from that of a native fibroin by at least 4% in terms of the absolute value of the sum of the differences in amino acid content of serine, glycine, and alanine; the cysteine ​​disulfide bonds between the fibroin heavy protein chain and the fibroin light protein chain of the fibroin-derived protein are reduced or eliminated; the serine content of the protein composition is reduced by 25% or more compared to native fibroin; Moreover, the serine content is at least about 5%; and the average molecular weight of the fibroin-derived protein composition is between 15 kDa and 36 kDa; (b) polysorbate-80 in an amount from about 0.02% to about 0.5% by weight; (c) one or more buffering agents; (d) one or more osmotic agents; A formulation comprising: wherein the formulation has a pH of 4.5 to 6.0, an osmolality of about 160 mOsm / kg to about 200 mOsm / kg, and a particle count of 50 / mL or less for particles having a diameter of 10 micrometers or greater after a storage period of more than 12 weeks at 4° C. to 40° C.

2. 2. The formulation of claim 1, wherein the protein composition comprises greater than 46.5% glycine amino acids, the protein composition comprises greater than 30.5% alanine amino acids, or a combination thereof.

3. 2. The formulation of claim 1, wherein the protein composition is reduced by more than 40% compared to native fibroin protein, such that the protein composition contains less than 8% serine amino acids.

4. 2. The formulation of claim 1, wherein more than 50% of the protein chains of the protein composition have a molecular weight within the range of 10 kDa to 40 kDa.

5. The formulation of claim 1, wherein the primary amino acid sequence of the fibroin-derived protein composition differs from that of native fibroin by at least 6% in terms of the difference in the sum of serine, glycine, and alanine content; the average molecular weight of the fibroin-derived protein is from about 15 kDa to about 30 kDa; and the pH of the formulation is between about 5.2 and about 5.

8.

6. 2. The formulation of claim 1, wherein the fibroin derived protein composition has an average molecular weight of about 15 kDa to about 25 kDa and the pH of the formulation is 5.3 to 5.

7.

7. 2. The formulation of claim 1, wherein the fibroin derived protein composition has an average molecular weight of about 18 kDa to about 25 kDa.

8. 2. The formulation of claim 1, wherein the weight percentage of fibroin derived protein is from about 0.05% to about 10%.

9. 2. The formulation of claim 1, wherein the buffer comprises histidine, acetate, citrate, glutamate, or a combination thereof.

10. 10. The formulation of claim 9, wherein the buffer concentration formed by the buffer is from about 10 millimolar to about 50 millimolar, or the concentration of each of the one or more osmotic agents in the formulation is from about 30 millimolar to about 40 millimolar.

11. 11. The formulation of claim 10, wherein the buffer comprises about 0.1% to about 1% sodium acetate by weight and about 0.01% to about 0.1% acetic acid by weight.

12. 10. The formulation of claim 1, wherein the osmotic agent comprises a simple sugar, an inorganic salt, or a combination thereof.

13. 13. The formulation of claim 12, wherein the osmotic agent comprises mannitol, dextrose, sodium chloride, magnesium chloride, or a combination thereof.

14. 14. The formulation of claim 13, wherein the osmotic agent comprises from about 0.10% to about 2% by weight dextrose and from about 0.10% to about 2% by weight magnesium chloride.

15. 10. The formulation of claim 1, wherein the formulation is stored in a container comprising glass or polyethylene, optionally the container being Type I borosilicate glass or LDPE.

16. (a) about 0.1% by weight to about 3% by weight of fibroin-derived protein, the primary amino acid sequence of which differs from that of native fibroin by at least 6% in terms of the absolute value of the sum of the differences in amino acid content of serine, glycine, and alanine; a fibroin-derived protein in which the cysteine ​​disulfide bonds between the fibroin heavy and light protein chains are reduced or eliminated; the fibroin-derived protein contains greater than 46% glycine amino acids and greater than 30% alanine amino acids; the serine content of the fibroin-derived protein is reduced by more than 40% compared to native fibroin protein, such that the fibroin-derived protein contains less than 8% serine amino acids; and the average molecular weight of the fibroin-derived protein is from about 15 kDa to about 35 kDa; (b) polysorbate-80 in an amount from about 0.02% to about 0.5% by weight; (c) about 10 mM to about 50 mM acetate buffer; and (d) one or more osmotic agents:

1. An aqueous formulation comprising: The formulation has a particle count of 50 / mL or less for particulates 10 micrometers or greater in diameter after a storage period of 12 weeks or greater at 4° C. to 40° C.; a pH of 5.2 to 5.8; and an osmolality of 175 mOsm / kg to 185 mOsm / kg.

17. 17. The formulation of claim 16, wherein the acetate buffer comprises from about 0.2% to about 0.3% by weight sodium acetate and from about 0.01% to about 0.03% by weight acetic acid.

18. 20. The formulation of claim 17, wherein the osmotic agent comprises about 0.6% to about 0.9% by weight dextrose and about 0.6% to about 0.9% by weight magnesium chloride hexahydrate.

19. 19. The formulation of claim 18, wherein the weight percentage of polysorbate-80 is from about 0.01% to about 0.1%.

20. 20. A combination according to any one of claims 1 to 19 for use in a method for treating an ocular disease comprising administering to a subject having the ocular disease an effective amount of the combination according to any one of claims 1 to 19, thereby treating the ocular disease.

21. 21. The formulation of claim 20, wherein the eye disease is dry eye syndrome.

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